Generation of insulin-producing cells from PDX1 and NKX6.1 gene-modified bone marrow mesenchymal stem cells

AIM: To differentiate bone marrow mesenchymal stem cells (BMSCs) into functional insulin-producing cells to produce sufficient pancreatic islet cells for transplantation. METHODS: Recombinant adenovirus vectors carrying PDX1 and NKX6.1 genes were constructed and the bone marrow mesenchymal stem cells were infected by the recombinant adenovirus combined with several cytokines for differentiation. The expressions of PDX1, NKX6.1 and insulin and C-peptide in the differentiated bone marrow mesenchymal stem cells were detected by RT-PCR and Western blotting. After the differentiated bone marrow mesenchymal stem cells were transplanted into subrenal capsule of diabetic mice, cell morphology of the grafts as well as their secretion of insulin and C-peptide were detected. Besides, regulating capacities of grafts on the blood glucose level of the diabetic mice were also detected. RESULTS: BMSCs induced by recombinant adenovirus (pAdxsi-CMV-PDX1/CMV-NKX6.1) and several cytokines showed positive dithizone staining and the expressions of β-cell related molecule such as insulin and glucose transporter 2 were detected by RT-PCR, which showed a sustaining and stable expression. The similar results were showed by Western blotting, immunohistochemical staining and indirect immunofluorescence. The insulin secretions in the cells stimulated with glucose at concentrations of 5.5 and 25 mmol/L in the experimental group were (1 240.4±109.3) mU/L and (3 539.8±245.1) mU/L, respectively, and were significantly higher than those in control group. Moreover, transplantation of the cells to STZ mice in treatment group made serum glucose recover to normal level. CONCLUSION: PDX1 and NKX6.1 gene-modified bone marrow mesenchymal stem cells differentiate into insulin-producing cells in vitro. When these cells transplanted into STZ induced diabetic mice, their serum glucose could return to the normal level and they could live well. Thus this is a promising method for diabetes treatment.     

Application of ESC-derived hepatic stem cells in therapeutic liver repo pulation

AIM: To study the proliferation, differentiation and the capacity of forming teratomas of ESC-derived hepatic stem cells in mouse pre-treated with retrorsine and 70% partial hepatotomy. METHODS: The ESC-derived hepatic stem cells, labelled with CFDA SE, were transplanted into BALB/c mouse liver. The distribution, incorperation and proliferation of transplanted cells were observed under fluorescent microscopy. Hepatic function was assayed by detecting albumin level in serum. The situation of forming teratomas in vivo was also evaluated.RESULTS: 1 week post-transplantation, some scattered region was green under fluorescent microscopy. The aera of green region increased apparently in 2 weeks, and cord-like structure was observed. Immunofluorescent staining of albumin demonstrated some positve cells, but there was no significant difference for albumin level in serum (P>0.05). No teratoma was formed in the experimental group, while a large teratoma was observed in control group in 6 weeks post-transplantation.CONCLUSION: The ESC-derived hepatic stem cells are normally incorporated into mouse liver parenchymal structure, proliferate and differentiate further in vivo and possess some hepatic functions without forming teratomas.     

Distribution and identification of immunocytes in humanized SCID mouse model

AIM: Humanized-NOD/SCID(hu-NOD/SCID) mouse model was established and the level of immune reconstitution was assessed in this model. METHODS: Mononuclear cells (MNC) and CD34⁺ cells were isolated or sorted from cord blood(CB). Human CD45, CD19, CD3 markers on cells from NOD/SCID murine peripheral blood(PB), bone marrow(BM), thymus were detected by FCM from 4 to 10 weeks after hematopoietic stem cell transplantation. After 10 weeks, the gene expressions of the human β₂M and RAG2 were detected by RT-PCR in PB or bone marrow of mice model. RESULTS: Human CD45, CD19, CD3 cells populations in PB and BM were found by flow cytometry in mice model transplanted with CD34+ cells or CB MNC from 4 to 10 weeks. The highest positivity of human lymphocytes was at 8 week after transplantation. The levels of human cell engraftment in mice transplanted with CD34⁺ cells were higher than those in mice transplanted with CB MNC. The mRNA of human β₂M and RAG2 were found by RT-PCR in BM.CONCLUSION: The higher level of human lymphocyte engraftment is established in NOD/SCID mouse model transplanted with CD34⁺ compared with CB MNC. The maturation of T lymphocytes could be happened in bone marrow of mice model.     

Inhibitory effect of Shenci capsule in combination with DDP on angiogenesis in mice with Lewis lung cancer

AIM: To investigate the inhibitory effect of Shenci capsule, a Chinese medicine, in combination with cisplatin (DDP) on the tumor growth and angiogenesis in mouse Lewis lung cancer.METHODS: Forty mice with Lewis lung cancer were randomly divided into 4 groups: Shenci capsule group, Shenci capsule in combination with DDP group, DDP group and control group. Shenci capsule, Shenci capsule in combination with DDP,DDP and sodium chloride wene given, respectively. After 21 days, the weight of subcutaneous tumors was measured, and the tumor inhibition rate was calculated. The microvessel density (MVD) and the protein level of vascular endothelial growth factor (VEGF) were detected by immunohistochemical(IHC) staining. The mRNA expression levels of kinase insert domain-containing receptor (KDR) and the receptor of VEGF were detected by FQ-PCR. RESULTS: The tumor growth inhibitory rate in Shenci capsule in combination with DDP group (63.99%) was higher than that in Shenci capsule group, DDP group and control group (P<0.01). The tumor MVD, VEGF protein and KDR mRNA expression level in Shenci capsule in combination with DDP group were lower than those in the above three groups (P<0.01). CONCLUSION: Shenci capsule in combination with DDP have addition or synergistic effects to inhibit the tumor growth and angiogenesis in mouse Lewis lung cancer. Down-regulation of VEGF and its receptor KDR may be one of the mechanisms that combined use of these two drugs for inhibiting angiogenesis.     

Signal pathways involved in reverse remodeling of the hypertrophic heart in rat after pressure unloading

AIM: Heterotopic transplantation of rat hypertrophic hearts has been shown to reverse chamber enlargement and regress cardiac myocyte hypertrophy. The purpose of this study was to gain a better understanding of molecular changes associated with the beneficial reverse remodeling after pressure unloading of hypertrophic heart. METHODS: Stable cardiac hypertrophy was induced by abdominal aortic constriction (AAC) in Lewis rats (6 weeks). Left ventricular (LV) pressure unloading was induced by heterotopic transplantation of hypertrophic hearts (AAC-HT) and/or normal hearts (NL-HT) (2 weeks). We measured heart weight (HW) and LV weight (LVW) of all groups. Cross-sectional area of cardiomyocyte was assessed by hematoxylin/eosin staining. We further analyzed the regulation of prohypertrophic signaling pathways of mitogen-activated protein kinases (MAPKs), Akt/GSK3β and NF-κB in both transplanted groups by Western blotting. RESULTS: The HW and LVW in AAC hearts were higher (P<0.05) than those in normal controls, but the transplanted hearts in AAC-HT group showed a significant reduction in HW and LVW compared to the AAC hearts. Pressure unloading induced a decrease in cardiomyocyte size in AAC-HT and NL-HT hearts. A significant decrease in phosphorylation of p44/p42 MAP kinases (ERK), Akt, GSK3β and NF-κB was detected in AAC-HT hearts, but the phosphorylation of p38 MAP kinase and Jun-N-terminal kinase (JNK) were not changed compared to AAC hearts. The phosphorylation of MAPKs, Akt/GSK3β and NF-κB showed no difference between NL-HT hearts and normal controls. CONCLUSION: Pressure unloading of the hypertrophic heart caused a reverse remodeling through regulating the ERK, Akt/GSK3β, and NF-κB signal pathways which act as potential target pathways for reversal of LV hypertrophy.     

Differentiation of Sca-1+ cells from murine fetal liver into renal cells in mice with acute renal failure

AIM: To study the effect of acute renal failure (ARF) on the differentiated frequency of Sca-1+ cells from murine fetal liver in irradiated mice. METHODS: The Sca-1+ cells from murine fetal liver were isolated with magnetic cell sorting (MACS) technique, the sex of which was identified by PCR. The 2×104 Sca-1+ cells were transplanted into a lethally irradiated (［60Co］, 8 Gy) inbred female mouse. After 8 weeks, these recipient mice were divided to A, B, and C groups at random (A group: irradiated; B group: ARF; C group: ARF and Sca-1+). The mice in B and C groups were induced to ARF with 50% (V/V) glycerin (11.6 mL/kg). 72 hours later, the mice in C group were injected with the fresh prepared Sca-1+ cells again. 8 weeks later, mice were sacrificed, and their kidneys were taken out, fixed and slices were prepared. Fluorescence in situ hybridization (FISH) of renal slices was performed and the pictures of them were taken and analyzed. RESULTS: The cells containing Y chromosome were found in renal slices from the mice in A, B and C groups, which located in epithelial cells of renal tubules, interstitium, glomeruli, and glomerular margin and increased gradually. The double and encircle zone of Y chromosome cells were found in the slices from the mice in B and C groups separately, which was consist of new renal tubules. The differentiation frequency of Sca-1+ cells in kidney in A, B and C groups were (1.65±0.18)%， (8.58±1.34)% and (18.13±1.91)%， respectively, which showed significant difference between former group and later group (P<0.01). CONCLUSION: The differentiation of Sca-1+ cells from murine fetal liver into renal cells and tissue is promoted by physiological microenvironment of acute damage and regeneration.     

Establishment of insulinoma animal models and observation of tumor characteristics

AIM：To establish the animal model of insulinoma and to analyze the properties of insulinoma for further study. METHODS：The hormone-releasing ability of rat insulinoma cell line INS-1 was detected in vitro. INS-1 cells were transplanted into the left kidney capsule of nude mice. The islets of the animals were destroyed by intraperitoneal injection of streptozocin (STZ) 3 d before transplantation or 2 weeks after transplantation. The venous blood was sampled, and the level of blood glucose less than 2.8 mmol/L was defined as successful establishment of insulinoma model. Different irritants were given to the model animals, and the changes of blood glucose and insulin content in serum were observed. The pancreatic tissues and the renal tissues in the injecting sites were taken from all mice for detecting insulin and glucagon by immunohistochemical staining. RESULTS：Insulinoma cells expressed insulin and glucagon at the same time. The blood glucose was less than 2.8 mmol/L 3 to 4 weeks after inoculation of INS-1 cells. Apparent tumor formed in the left kidneys where INS-1 cells were transplanted and the tumor diameters were more than 1 cm. The level of blood glucose transiently increased to higher than the normal level in the mice with tumor cell transplantation after intraperitoneal injection of STZ, and then decreased gradually and returned to less than 2.8 mmol/L after 2 weeks. The level of blood glucose in the normal nude mice after administration of STZ increased significantly. After transplantation of INS-1 cells, the level of blood glucose decreased gradually, and returned to less than 2.8 mmol/L after 4 weeks. After stimulated with high glucose, the blood glucose levels in the mice with 3 methods to establish the insulinoma models showed lower glucose peaks than that in the normal control mice. After stimulated with high glucose plus arginine or acetylcholine in the normal animals, the blood glucose peak was lower than that in the normal animals only stimulated with high glucose, and rapidly recovered to the normal level. However, the levels of blood glucose in the mice with 3 methods to establish the insulinoma models under the same stimulations were significantly higher than that in the mice only stimulated with high glucose. After stimulated with high glucose plus norepinephrine, the blood glucose peak time in the normal animals delayed, and the blood glucose level declined slowly. After stimulated with high glucose plus norepinephrine, the levels of blood glucose in the mice with 3 methods to establish the insulinoma models increased as compared with that in the mice only stimulated with high glucose. Compared with normal control group, serum insulin in insulinoma mice increased significantly. CONCLUSION：The insulinoma animal model is successfully established by transplantation of INS-1 cells into the renal capsule of nude mice. The insulinoma cells express both insulin and glucagon, and are not easily damaged by STZ.     

Establishment of human pancreatic tumor xenograft mouse model for evaluating tumor-homing and gene-silencing effects of siRNA-loading nanoparticles

AIM：To establish an effective and rapid method to develop transplanted subcutaneous pancreatic carcinoma by inducing PANC-1 cells into nude mice, and then use this mouse model to evaluate the tumor-homing and gene-silencing effects of siRNA-loading nanoparticles in vivo. METHODS：Different numbers of PANC-1 cells in 100 μL or 300 μL PBS were inoculated subcutaneously into the right flank of BALB/c (nu/nu) mice. When the tumor volume reached 100 mm3, siRNACY5.5 nanoparticles were injected through the mouse tail vein to perform in vivo imaging assay. Besides, the mice were randomly divided into 3 treatment groups treated with PBS, scrambled control RNA nanoparticles and siKras nanoparticles, respectively. The protein expression of Kras was detected by Western blotting and immunohistochemical staining. RESULTS：After inoculated with 1×107 PANC-1 cells in 300 μL PBS, all mice developed tumors within 2 weeks. The in vivo results showed that siRNA-loading nanoparticles accumulated in the tumor tissues and exerted gene silencing effect. CONCLUSION:In the present study, an effective and rapid method was established for PANC-1 cells to induce transplanted subcutaneous pancreatic carcinoma in nude mice within 2 weeks, which is suitable for in vivo imaging and treatment evaluations as a reproducible and reliable way for the further experiments.     

Human T-cells developed in a triple chimera animal model and tolerant to porcine xenoantigens

AIM: To determine the characteristics (including stability) of a triple chimera animal model generated by implantation of human fetal thymus/liver (Thy/Liv) fragments, fetal liver hematopoietic cells, and porcine bone marrow cells (BMCs) in NOD/SCID mice. METHODS: NOD/SCID mice were treated with 3 Gy of whole-body irradiation (WBI) and divided into three groups. Group A was given intravenous porcine BMCs. Group B was given an implant of human fetal Thy/Liv fragments (1-2 mm3) under the right kidney capsule and additionally an intravenous injection of human fetal liver mononuclear cells (MNCs) purified from the fetal livers. The treatment of group C was the combination of both group A and B. Three weeks after Thy/Liv transplantation and every three weeks thereafter, peripheral blood was drawn to determine the level of porcine and human chimerism using multicolor flow cytometric (FCM) analysis. The proliferation of the T cells was tested in a standard mixed lymphocyte reaction (MLR). RESULTS: Porcine chimerism persisted (>20 weeks) in all mice that received porcine BMCs, including mice that underwent human Thy/Liv implantation later. Human chimerism persisted >20 weeks in all mice that received Thy/Liv implants, regardless of whether they received porcine BMCs transplants. CONCLUSION: Human immune system is reconstituted by co-transplantation of human fetal Thy/Liv and human fetal liver MNCs in NOD/SCID mice with porcine hematopoietic chimerism. The human T cells are immunocompetent and tolerant to donor porcine BMCs.     

Role of VEGF in establishment of Walker-256 transplanted liver cancer model in SD rats

AIM：To evaluate the safety and feasibility of using vascular endothelial growth factor (VEGF) in the establishment of Walker-256 transplanted liver cancer model. METHODS：SD rats (n=45) were divided into 3 groups:via the caudal vein, the rats in normal saline (NS) group were injected with 0.9% sodium chloride (0.1 mL/d), the rats in 20 mg/L VEGF group were injected with 20 mg/L VEGF (0.1 mL/d), and the rats in 40 mg/L VEGF group were injected with 40 mg/L VEGF (0.1 mL/d). All the injection began 1 week before transplantation of liver cancer, and stopped on the day the cancer model was established. Prepared tumor tissue was transplanted into the subcapsular space of the liver. Three days, 1 week and 2 weeks after the transplantation, magnetic resonance imaging (MRI) was performed for analyzing the tumor growth and the characteristics. The overall survival of the rats was also recorded. RESULTS：Successful establishment of Walker-256 transplanted liver cancer model was achieved. Among 45 rats, 1 rat died 1 d after implanting the tumor both in NS group and 20 mg/L VEGF group, while 3 rats died in 40 mg/L VEGF group 1 week after building the model, mainly because of the progression of tumors. Three days after modeling,the numbers of the rats in which the tumor was positively detected by MRI in 3 groups were 0, 7 and 10, respectively; 1 week after modeling, those were 3, 13 and 13, respectively; 2 weeks after modeling,those were 12, 13 and 10, respectively. Between NS group and 20 mg/L VEGF group, the statistical significance existed in the number of the rats in which the tumor was positively detected by MRI after 3 d of implanting, so did the NS group and 40 mg/L VEGF group. No statistical significance in the overall survival time between NS group and 20 mg/L VEGF group (P＞0.05) was observed, but the significance existed between 40 mg/L VEGF group and NS group (P<0.01). CfONCLUSION:The application of VEGF at dose of 20 mg/L and 0.1 mL/d shortens the time to establish the transplanted liver cancer model without influence on the overall survival, which is a safe, feasible and efficient way, and is more suitable for anti-VEGF drug investigation.     

Differential expressions of ameloblastoma related genes determined by microarray

AIM: This study aims to screen the differentially expressed genes related to the pathogenesis of ameloblastoma by cDNA microarray. METHODS: The total RNAs were isolated from ameloblastoma and tooth germ, respectively. The RNAs were purified by oligotex. Both the mRNAs from two kinds of tissues were reversely transcribed to cDNA with the incorporation of fluorescent-labelled dUTP to prepare the hybridization probes. The mixed probes were hybridized to cDNA microarray. Tumor- related genes were screened through the analysis of fluroescent intensity. RESULTS: 722 genes exhibited significant changes in expression levels in the ameloblastoma in comparison with tooth germs tissues. 240 genes were overexpressed more than doubled (92 genes were more than 3-fold), and 482 genes were underexpressed to below 0.5 of the control level. CONCLUSION: Microarray technique facilitates large scale and rapid identification of potential target genes of ameloblastoma.     

Purification and differentiation potentiality of fetal liver mesenchymal stem cells from mouse

AIM: To purify and investigate the differentiation potentials of fetal liver mesenchymal stem cells (flMSCs) from murine in vitro.METHODS: flMSCs from mouse fetuses at embryonic and fetal day (ED)13.5 or ED14.5 were isolated by adhering to plastic and passaged by modified method.Cell cycle and phenotype were analyzed by flow cytometry.The cell differentiation was induced by special induction media.The cells differentiated to adipose,cartilaginous and osteoid tissues were identified with oil red O,Toluid blue,alkaline phosphatease (ALP) and von Kossa’s staining.The cells differentiated to neural-like cells were detected by RT-PCR and immuno-staining.RESULTS: Fibroblast-like cells predominated in culture.(83.76±2.88)% of flMSCs stayed in the G₀/G₁ phases.Homogenous cells were positive for mesenchymal lineage markers CD44,CD29,but not for markers of hematopoietic cells CD45,CD11b.flMSCs were able to differentiate into adipogenic,chondrogenic,osteogenic and neurogenic cells.CONCLUSION: flMSCs can be purified by modified plastic-attachment method and have multiple differentiation,which is available to stem cell therapy for various diseases.     

Effects of RNA interference of FABP5 on subcutaneous tumor of human hepatocellular carcinoma cell line HepG 2 transplanted in nude mice

AIM: To investigate the effect of recombinant lentiviral vector for RNA interference (RNAi) on the expression of fatty acid-binding protein 5 (FABP5) gene in hepatocellular carcinoma HepG2 cells and tumor formation in nude mice.METHODS: RNAi lentiviral vector was used in the experiment. Human hepatocellular carcinoma HepG2 cells were divided into 3 groups:the HepG2 cells in experimental group were transfected with the recombinant lentivirirus vector LV-shRNA-FABP5, the cells in negative control group were transfected with a control lentiviral vector LV-shRNA-NC, and the cells in normal control group were without any treatment. The nude mice were randomly divided into 3 groups. The growth of the transplanted tumor cells in the nude mice was observed. The tumor growth curve, volume and weight were determined 4 weeks after the cell inoculation. The expression of FABP5 was detected by real-time PCR, Western blot and immunohistochemical staining.RESULTS: Transfection of the lentiviral vector FABP5-shRNA obviously reduced FABP5 expression in the HepG2 cells. Tumor formation was all positive in the 3 groups of the nude mice inoculated with the tumor cells. Compared with normal control group and negative control group, the tumor growth slowed significantly in experimental group with smaller volume and weight. FABP5 expression in the transplanted tumor tissues was significantly down-regulated at mRNA and protein levels in experimental group as compared with normal control group and negative control group.CONCLUSION: RNAi-induced down-regulation of FABP5 effectively inhibits the growth of transplanted hepatocellular carcinoma, suggesting that FABP5 gene may be an effective target for gene therapy in treating liver cancer.     

Effects of edaravone on expression of caspase-3 and caspase-12 in juvenile rat hippocampus after status convulsion

AIM: To investigate the effect of edaravone (ED) on the expression of caspase-3 and caspase-12 in the juvenile rat hippocampus after status convulsion (SC).METHODS: Juvenile male Sprague-Dawley rats were randomly divided into normal saline (NS) control group, SC group and ED treatment group. The rats in each group were further divided into 5 subgroups according to different time points. The rats in SC group were kindled into epilepsy by lithium-pilocarpine chemical method. The protein expression of caspase-3 and caspase-12 was determined by immunohistochemistry methods. The mRNA expression of caspase-3 and caspase-12 was detected by RT-PCR. RESULTS: (1) The IA value of caspase-3 positive cells in 24~72 h SC group increased compared with NS group. With ED intervention, the IA value of caspase-3 positive cells decreased as compared with 48~72 h SC group. The results of RT-PCR showed that the mRNA expression of caspase-3 was similar to the changes of protein. (2) The results of immunohistochemistry showed that the IA value of caspase-12 positive cells in 12~72 h SC group increased compared with NS group. With ED intervention, the IA value of caspase-12 positive cells decreased as compared with 24~72 h SC group. The results of RT-PCR showed that the mRNA expression of caspase-12 was similar to the changes of protein. (3) In ED group, Ⅴ grade convulsion was lower than that in SC group, and the latent period of seizures in ED group was significantly longer than that in SC group. CONCLUSION: Edaravone inhibits the expression of caspase-3 and caspase-12 in pilocarpine-induced seizures in rat hippocampus, suggesting that edaravone has protective effect against the damage caused by status convulsion.     

NK cells from donor liver inhibit NF-κB activity and IL-15 expression after liver transplantation in rats

AIM:To investigate the mechanism that donor liver natural killer (NK) cells alleviate acute rejection after liver transplantation by observing the secretion level of interleukin 15 (IL-15) in peripheral blood, the protein expression of IL-15 in transplanted liver tissues and the activity of NF-κB in spleen tissues in rat acute liver graft rejection model. METHODS:An acute rejection model of liver transplantation in rats was established by the modified two-cuff method, in which Lewis rats were used as donors and BN rats as recipients. The donor leukocytes were depleted by whole body irradiation of ［60Co］ source and the donor liver immunity was reconstituted by transfusion of liver NK cells from the same type of donor (donor type liver NK cells, dtlNKs) via portal vein immediately after grafting the irradiated liver. The rats were divided into the following groups: group A, acute rejection group; group B, BN rats receiving the liver of Lewis rats with ［60Co］ irradiation; group C, BN rats receiving the liver of ［60Co］-irradiated Lewis rats and treated with dtlNKs via the portal vein. The recipients were sacrificed at 1 d, 3 d and 7 d after transplantation. IL-15 level in peripheral blood was detected by ELISA. The expression of IL-15 in the liver grafts was determined by Western blotting. NF-κB activity in the spleen tissues of recipient rats was identified by electrophoretic mobility shift assay. The survival quality and living time in crude survival subgroup were observed. RESULTS:Acute rejection in group B was severer than that in group A and group C. The rats in group B showed significantly shorter average survival time compared with group A and group C. At 3 d and 7 d after transplantation, the IL-15 content in peripheral blood was significantly higher in group B than that in group A and group C. The expression of IL-15 in transplanted liver tissues was significantly higher in group B than that in group A and group C. The activity of NF-κB in the spleen tissues was higher in group B. CONCLUSION: IL-15 might be a significant indicator for monitoring acute rejection after liver transplantation. The donor liver NK cells modulate the immunity of liver transplantation by inhibiting the expression of IL-15 via the suppression of NF-κB activity.     

Role of bcl-2 gene expression in inhabition of hepatocellular carcinoma by genistein in nude mice

AIM:To probe into the role of 1, 4, 5 - trisphosphate inositol (IP3) and bcl-2 gene expression in inhibiting hepatocellular carcinoma of nude mice by genistein. METHODS:Animals with hepatocellular carcinoma were treated with genistein 1 mg·kg-1·d-1 (ip) for 3 weeks. The volume and weight of tumaor were measured. IP3, bcl-2 mRNA, Bcl-2 protein were assayed by IP3-［3H］ Birtrak assay, RT-PCR, Western blotting, respectively. RESULTS:The tumor volume and weight of animals treated with genistein were lower than those in control (42.7mm3±27.8mm3 vs 52.3mm3±26.5mm3, 42.7mg±27.8 mg vs 91.3mg±31.4 mg). IP3 content was lower than that in control ［(13.4±1.4)nmol/g protein vs (35.3±6.6)nmol/g protein］. bcl-2 mRNA expression was lower in group treated with genistein than that in control (RI which was the gray degree multiply area of bcl-2 / the gray degree multiply area of β-actin 0.48±0.02 vs 0.56±0.15). Bcl-2 protein expression was lower in group treated with genistein than that in control (RI 1.69±0.52 vs 1.37±0.48). CONCLUSION:Genistein inhibits growth of transplanted hepatocellular carcinoma in nude mouse liver by reducing IP3 production and down-regulating bcl-2 gene expression.     

Comparative study on intraocular transplatation of three B16 melanoma cell lines in mice

AIM:To establish an animal model for studying the development and metastasis of melanoma.METHODS:C57BL/6 mice were used as host to receive melanoma cell transplantation. Three kinds of melanoma cell lines, B16F0, B16F1 and B16F10, cultured to prepare the cell suspensions, were transplanted into the mouse anterior chamber (AC) of the eye. The time of eyeball diabross, time of survival and metastasis of lymph node and lung were observed.RESULTS:The time of eyeball diabross in F10 group was earlier than that in other groups. The time of eyeball diabross was no difference between F0 and F1 groups. Metastasis was developed 18 days after transplantation in F1 and F10 groups, where the tumor cells was found in ipsilateral cervical lymph nodes. The melanoma cells metastasized to lung in all three groups 28 days after transplantation. The survival time in F0 group was longer than F1 and F10 groups. There was no difference in survival times between F1 and F10 group.CONCLUSION:The differences of three kinds of melanoma cell lines in tumor development and metastasis provided the evidence that was useful for choosing suitable animal model further to study the eye melanima.     

Application of anti-CD20 antibody in hematopoietic stem cell transplantation for sensitized recipients

AIM: To find a strategy for enhancing engraftment of hematopoietic stem cell in sensitized recipients and to study the effects of anti-CD20 antibody in hematopoietic stem cell transplantation. METHODS: BALB/c mice were sensitized by transfusions of allogeneic spleen cells on 14 d and 7 d. Anti-CD20 antibody (2 mg/mouse) was intravenously injected into sensitized recipients on 11 d. The recipients were used as experimental group, while RPMI-1640 medium (0.2 mL/mouse) was used as control. The sera and splenocytes obtained from the recipients were tested for donor reactive antibody and CD19+ B cells on 0 d. In addition, the recipients were transplanted with 1×107 C57BL/6 bone marrow cells after lethal irradiation on 0 d. The survival rates were observed and blood counts were studied post transplantation. RESULTS: The cytotoxic index in the experimental group and control group were (37.00±3.46)% and (51.80±3.49)%, respectively, and the differences were significant (P<0.01). The percentages of CD19+ B cells in experimental group and control group were (17.32±3.02)% and (34.26±2.87)%, respectively, and the differences were significant (P<0.01). All the recipients in both experimental group and control group died about 14 d post transplantation. The median time was 13 d and 11 d in experimental group and control group, respectively, and no significant difference was found between these two groups (P>0.05). Moreover, a rapid disappearance was observed in the white blood counts, hemoglobin, and platelet of dying animals, indicating the animals died from hematopoietic failure. CONCLUSION: Anti-CD20 antibody is able to deplete B cells and reduce the level of antibody in sensitized recipients, but it can’t enhance the engraftment of allogeneic hematopoietic stem cells in the sensitized recipients.     

Expression of oxygenase-1 protein and mRNA on mouse acute viral myocarditis caused by coxsackie viruses B3

AIM: To observe the dynamic variation of oxygenase-1 protein and mRNA on mouse acute viral myocarditis caused by coxsackie viruses B3.METHODS: A total 72 inbred male BALB/c mice of 4-6 weeks were divided randomly into 2 groups as follows: 32 mice were inoculated intraperitoneally (ip) with virus free 1640 culture solution 0.1 mL on day 0 as blank group (C); 40 mice were ip 0.1 mL tissue culture infectious dose 50 (TCID50 is 10-4.36/mL) coxsackie viruses B3 (CVB3) on day 0 as VMC group (V), then each mouse in both groups was ip 0.1 mL NS every day. 8 mice in each of C group and V group were sacrificed on 4, 8, 15, and 21 d respectively after infections. The blood specimens gathered by taking out the eyeballs of mice were tested for the content of carboxyhemoglobin (COHb) using spectrophotometer method.The heart tissue slides were also stained by immunohistochemistry (IHC) for HO-1 and in situ hybridization (ISH) for HO-1 mRNA. The histological and ultrastructure changes were observed under light microscope and electron microscope.RESULTS: (1)The histopathological changes of myocardial cells: many inflammatory cells were found in the heart and large area myocardial cells necrosis was observed under light microscope. The inflammatory area was reduced at late stage in the heart of mouse in group V, while the myocardium in group C was normal. (2) The myocardial observation by electron microscopy: the myofibril in group V was dissolved and mitochondrial membrane disappeared, mononuclear cell infiltration was also observed under electron microscopy, which contained many lysosomes. The myocardial cells in group C were normal. (3) The changes of blood COHb level: compared with group C, the group V COHb level showed significantly higher on the day 8 and day 15 after CVB3 innoculation (0.047±0.005 vs 0.031±0.004; 0.076±0.006 vs 0.030±0.005, P<0.01). No obvious change in group C was observed. (4) The result of HO-1 IHC staining: myocardial cells had positive expression in group V, group C was negative. The absorbance (A) values in group V was significantly higher than that in group C (P<0.01) at different time points. (5) The result of HO-1 ISH was similar to HO-1 IHC. The A values in group V was all higher than that in group C (P<0.01).CONCLUSION: Viral myocarditis caused by coxsackie viruses B3 induces the expression of HO-1 mRNA and protein, and these expressions may play self-protection in inflammation injury in myocardial cells.