Effects of blocking the expression of PKARⅠβ gene with antisense nucleotide on Shuang long-Jie gu Pill (SLJGP) containing serum osteoblast proliferation

AIM: To further investigate the role of PKARⅠβ in the growth-promoting effects of shuang long Jiegu pill (SLJGP), a Chinese medicine, on cultured osteoblasts. METHODS: pcDNA- antiPKARⅠβ, a recombinant expressing the antisense sequence of PKARⅠβ, was constructed and transformed HFOB1.19 by lipofectin. MTT was undertaken to assess the cell growth with the treatment of high dosage of SLJGP containing serum. RESULTS: Antisense gene blocked the growth-promoting effects of SLJGP containing serum on HFOB1.19. CONCLUSION: The function of SLJGP is closely related to cAMP-dependent protein kinase A.     

牛精子冷冻损伤研究进展

人工授精技术是目前通过冷冻精液进行牛遗传改良而应用最为广泛的生物技术。虽然如此,由于精液产品的质量而导致人工授精的遗传影响被限制,在精液的冷冻－解冻过程中40%~50%的活精子失去完整性或功能受到损伤。作者在参阅国内外相关文献的基础上,通过阐述冷冻－解冻对精子细胞的损伤作用及机理、精浆对牛冷冻精液质量的影响、冷冻－解冻过程中精子的过氧化损伤,以及冷冻－解冻后精子相关基因与蛋白质的变化等,以期为牛高质量冷冻精液的研究提供一定参考,进一步提升冷冻精液质量。     

The transdifferentiation of Sca-1+ cells from murine fetal liver

AIM:To explore transdifferentiation potential of Sca-1⁺ cells from murine fetal liver. METHODS:2×10³ of Sca-1⁺ cells from male murine fetal liver were transfused into female mouse irradiated lethally with γ ray from ⁶⁰ Co source (10 Gy) via tail vein. Two months later, FISH and immunohistochemistry were used to detect the situation for transdifferentiating of the donor cells (male cells) in tissues of female recipient mouse. RESULTS:The renal tubular epitheliocyte-like and neurocyte-like cells with Y chromosome were found on the sections of renal and brain tissues from female recipient mice. These cells have phenotype characteristics of RCA⁺/CD₄₅^-F_4/80^- and NueN⁺/CD₄₅^-F_4/80^-, respectively. CONCLUSION:The evidence is provided for Sca-1⁺ cells from murine fetal liver to transdifferentiate into both renal and brain tissue cells.     

发酵床养猪垫料中病原防控的研究

为了有效防控发酵床垫料中霉菌、寄生虫、大肠杆菌及金黄色葡萄球菌等的生长,选取了几十种中药及化学制剂进行了体外抑菌试验。结果表明,所有化学药物对垫料中一种或几种有益菌有不同程度的抑制效果;蒲公英、鱼腥草、茵陈、秦皮、大青叶、淫羊藿、青蒿、穿心莲、龙葵、博落回、地锦草对垫料中有益菌无抑制作用,对大肠杆菌及金黄色葡萄球菌有一定的抑制效果;丙酸钙对有益菌无抑制作用,对霉菌有明显的抑制效果;贯众与土荆皮对有益菌无抑制作用,可作为垫料中的驱虫药。     

Induction of tumor-specific cytotoxic T lymphocytes in vitro by dendritic cells pulsed with different glioma antigens

AIM: The goal of this study was to compare different methods for tumor antigen preparation, to observe the induction of tumor-specific cytotoxic T lymphocytes in rats by dendritic cells (DCs) pulsed with different tumor antigens. METHODS: The precursors of dendritic cells were isolated from bone marrow of rats, stimulated in vitro with recombinent rat granulocyte-macrophage colony-stimulating factor (rrGM-CSF) and interleukin-4 (rrIL-4). Then rat DCs were pulsed with C6 tumor cell antigens prepared with different methods: freeze-thaw, boiling or total protein extracted from ultrasonic crushed tumor cell. Subsequently primed DCs were cocultured with T lymphocytes isolated from spleen to induce CTL. Lymphocyte chemoattractant factor from DCs and cytokine IFN-γ release were determined by ELISA, the cytotoxicity of CTL was assayed by JAM test. RESULTS: DCs pulsed with boiled tumor cell in vitro induced an enhanced ability of T-cell proliferation and cytotoxic T lymphocyte activity.CONCLUSION: Our results demonstrated that DCs primed with boiled tumor cell may represent a method for inducing immune responses against the entire repertoire of tumor antigens of malignancies.     

Expression of phenylalanine ammonia-lyase cDNA from rice in E. coli BL21DE3

AIM: To study the expression and its kinetics of rice phenylalanine ammonia-lyase gene encoding into E. coli as the basis of treatment for phenylketouria. METHODS: The phenylalanine ammonia-lyase-1-cDNA(rPAL-1-cDNA) from rice was recombined into E. coli high expression vector pET-28c and transformed into E. coli host strain BL21DE3. Engineering bacteria was then inducted by isopropyl-β-D-thiogalactoside (IPTG) for 1, 3, 5, 7 hours, in order to obtain high level expression. RESULTS: After induction, the expression level of fusion protein was 21.40%, 30.60%, 35.40%, 35.43% respectively. The fusion protein exhibited a band of 78.6 kD on SDS-PAGE analysis, but was not found in controls.The target protein was mainly existed in the form of inclusion body. CONCLUSION:Rice PAL gene expressing E. coli was established by gentic engineering technique.     

Study on relationship between expression of PKCα in glomeruli and development of nephropathy in diabetic rats

AIM:To observe the dynamic changes of expression of PKCα, TGF-β₁ and α-SMA in glomeruli of diabetic rats induced by the alloxon and to invesitigate their roles in the diabetic nephropathy(DN).METHODS:Rats were randomly divided into four groups: normal control group (group A), diabetic group of one week (group B), diabetic group of one month (group C), diabetic group of two months (group D). Immunohistochemistry and Western blotting were used to detect the expression of PKCα, TGF-β₁ and α-SMA in renal tissue of all groups. Blood glucose, triglycerides, cholesterol, creatinine and urine protein were analysed by chemical methods. The morphological changes of renal tissue were checked through microscopy.RESULTS:The expression of PKCα and TGF-β₁ in renal tissue of diabetic groups were increased comparing with those of nomal control group(P<0.05). The mesangial cells expressed α-SMA in two months group. Chronologically the expression of PKCα, TGF-β₁ and α-SMA were positively correlative with each other and the impairment of kidney was also observed.CONCLUSIONS: During the DN process the expression of PKCα increased. PKCα raised GFR and the permeability of glomerular filtration membrane which enhanced urinary albumin excretion. PKCα also increased expression of TGF-β and therefore to induce the expression of α-SMA. The appearance of α-SMA was a marker of the phenotypic transform of renal cells.     

Influence of irbesarten on renal hypertrophy in streptozotocin-induced diabetic rats LIU Bi-cheng,LUO Dong-dong,ZHANG Xiao-liang.

AIM: To investigate the influence of irbesartan (Irb), a new angiotensin II receptor 1 antagonist, on renal hypertrophy in streptozotocin (STZ)-induced diabetic rats. METHODS: Sprague-Dawley (SD) rats were randomly divided into three groups: normal group (Group N, n=7), diabetic group (Group DN, n=6) and irbesartan treated group (Group DNI, n=7). In the experimental group, after the rats subjected to uninephrectomy, STZ was given by peritoneally injection at bolus dose of 50 mg/kg to induce diabetes. Blood glucose (BG), body weight (BW), urinary albumin excretion (Ualb), 24 hour proteinuria (24 h Upro) were measured at week 4, 8, 12, respectively. By the end of experiment at week 12, creatinine clearance (Ccr), kidney weight (KW), indicator of renal hypertrophy (KW/BW), renal total protein content (RTP), glomerular area (AG) and glomerular volume (VG) as well as glomerular basement membrane (GBM) were determined by semi-quantitative pathology technique. RESULTS: It was showed that there was no significant difference in BG between group DN and DNI, while Irb significantly reduced the increasing of Ualb, 24 h Upro in diabetic rats compared to control group (P＜0.01, respectively). Furthermore, Irb markedly inhibited the increasing of KW, KW/BW, RTP, A_G, V_G in diabetic rats (P＜0.05, P＜0.01, respectively). Of interest, Irb significantly prevented the increasing of GBM in diabetic rats. CONCLUSION: Irb exerts its early renal protective action by reducing proteinuria and inhibiting renal hypertrophy as well as the thickening of GBM.     

Effect of compound salvae-dropping-pill on intracellular free calcium concentration of cultured rat myocardial cells in case of hypoxia and reoxygenation

AIM:To examine the effect of compound salvae-dropping-pill (CSDP) on intracellular free calcium in cultured rat myocardial cells subjected to hypoxia and reoxygenation.METHODS:The Fluo- 3/AM was applied to probe intracellular calcium concentration and the fluorescent intensity was detected using laser confocal microscopy technique.RESULTS:Fluorescent intensity in hypoxia plus CSDP group was significantly lower (1 217.78±312.07) than that of hypoxia group (1 509±508.48), and the Fluorescent intensity of hypoxia/reoxygenation plus CSDP group was also markedly lower (1 567.91±577.61) than that of hypoxia/reoxygenation group (1 617.60±477.53).CONCLUSION:The cultured rat myocardial cells could be effectively protected by administration of CSDP in case of hypoxia and reoxygenation through decreasing the intracellular calcium concentration.     

Inhibition of K562 cell growth by antisense drug targeting with VEGF mRNA in vitro

AIM: To investigate inhibition of K562 cell growth by antisense drug targeted VEGF mRNA. METHODS: X7, 20-mer antisense sequences were selected, synthesized and modified with phosphorothioate. The drug was transfected into K562 cells in the present of lipofection. Cell growth was assayed by trypan blue dye exclusion assay and MTT. The level of VEGF protein in the media was determined by ELISA. The morphology of apoptotic cells were observed by Giemsa staining, and the propotion of apoptotic cells was detected by flow cytometry. RESULTS: The antisense drug inhibited growth of K562 and downregulated expression of VEGF protein significantly, compared with Scrambed control group and showed dose-dependent relation. Signs of apoptosis of K562 cells were not observed. CONCLUSION: Inhibition of K562 cell proliferation, but not cells apoptosis induction is the mechanism of inhibing growth of K562 cells by antisense drug targeted VEGF mRNA. At same time, VEGF has function of promoting K562 cell proliferation, and VEGF mRNA may be a new target attached by drugs.