猪骨髓间充质干细胞分离培养及生物学特性

采集健康猪股骨骨髓,利用percoll梯度离心法分离单个核细胞,进行体外原代和传代培养,并用不同代数细胞进行了细胞生长能力、膜表面抗原(CD105、CD90、CD45)及诱导分化能力的检测.结果显示分离培养的单个核细胞贴壁生长,形态呈成纤维细胞样及涡旋状克隆团；细胞传代至第17代生长状况仍然良好；用F3代细胞进行膜表面抗原标记显示,CD90和CD105阳性表达率分别为(97.4±1.8)％和(99.6±0.9)％,而CD45阳性表达率仅为(1.8±0.55)％,证实这些细胞具有猪骨髓间充质干细胞(Mesenchymal stem Cells,MSCs)表面抗原；经地塞米松、维生素C和β-磷酸甘油等诱导F3代细胞,21d后出现钙物质沉积细胞群,用茜素红和Von kossa染色呈阳性,证实这些细胞已分化形成成骨细胞；经地塞米松、IBMX、胰岛素及吲哚美辛等诱导F3代细胞,21d后细胞质出现脂滴样结构,用油红O染色呈阳性,证实已分化形成成脂细胞；冷冻-解冻细胞的膜表面抗原及多能分化能力与未冻存细胞的检测结果基本一致.结果证实,从猪骨髓中分离培养及经传代扩增获得的贴壁细胞是纯化的MSCs.     

Effects of L-carnosine on NIT-1 islet cells

AIM: To investigate the protective effect of L-carnosine on insulin secretion, proliferation and apoptosis of β-cells impaired by high glucose. METHODS: NIT-1 cells were pre-treated with glucose at concentrations of 11.1 mmol/L (control level) and 33.3 mmol/L (high level) for 72 h, and then the cells were stimulated with various concentrations of glucose (0, 5 and 25 mmol/L) and/or L-carnosine (0, 1 and 20 mmol/L). The level of insulin in the medium was measured by radioimmunoassay. To detect the effect of L-carnosine on proliferation and apoptosis, NIT-1 cells were divided into 4 groups according to different culture conditions for 72 h: group C (with 11.1 mmol/L glucose), group H (with 33.3 mmol/L glucose), group H+A (with 33.3 mmol/L glucose+ 1 mmol/L L-carnosine) and group H+B (with 33.3 mmol/L glucose +20 mmol/L L-carnosine). Proliferous or apoptotic cells were identified by BrdU labeling and flow cytometry (labeling with annexin V-FITC/PI),respectively. Total RNA was extracted and the mRNA expression of caspase-3 and bcl-2 was measured by RT-PCR. The caspase-3 activity was also checked by fluorometric assay kit. RESULTS: The insulin in high-level glucose group was lower than that in control-level glucose group. L-carnosine at concentration of 20 mmol/L notably increased the insulin secretion of the cells pre-treated with glucose at control level or high level. The proliferation and apoptosis were both increased in group H compared with group C, but the total cell counts declined because the apoptotic rate was higher than the proliferation rate. L-carnosine at concentration of 1 mmol/L significantly increased the proliferation rate and decreased the apoptotic rate. The mRNA level of caspase-3 was decreased and the mRNA level of bcl-2 was increased after the cells were treated with L-carnosine at concentration of 1 mmol/L. L-carnosine at concentrations of both 1 mmol/L and 20 mmol/L significantly decreased the caspase-3 activity. CONCLUSION: L-carnosine at high level directly stimulates insulin secretion in NIT-1 cells, and L-carnosine at normal level promotes the cell proliferation and inhibits apoptosis induced by high concentration of glucose. Caspsase-3 and Bcl-2 may be partly involved in this process.     

Effects of human bone morphogenetic protein 2 and 9 on proliferation,apoptosis and migration of human gastric carcinoma MNK-45 cells

AIM: To investigate the effects of human bone morphogenetic protein 2 (BMP2) and BMP9 on the proliferation, apoptosis and migration of human gastric carcinoma cell line MNK-45. METHODS: Immunocytochemical staining, MTT assay, wound-healing test, Transwells migration test, Hoechst 33258 staining and flow cytometry (FCM) were used to determine the infection of AdBMP2 and AdBMP9 on the proliferation, apoptosis and migration of MNK-45 cells. The expression of GSK-3β (including p-GSK-3β and total GSK-3β) and β-catenin in MNK-45 cells was also detected by Western blotting. RESULTS: The proliferation of MNK-45 cells was inhibited from the third day on and in a time-dependent manner after infected with AdBMP2 and AdBMP9. The results of Hoechst 33258 staining and FCM proved that apoptosis rates in BMP2 group and BMP9 group were higher than that in GFP group. Both wound-healing test and Transwell experiment indicated that up-regulating the expression of BMP2 and BMP9 inhibited the migration of MNK-45 cells. The phosphorylation levels of GSK-3β in BMP2 group and BMP9 group were higher than that in GFP group. However, no significant change of β-catenin among groups was observed. CONCLUSION: Up-regulation of BMP2 and BMP9 expression inhibits the proliferation of MNK-45 cells.     

PPARδ agonist GW501516 inhibits ox-LDL-or high glucose-induced apoptosis in human umbilical vein endothelial cells

AIM: To investigate the inhibitory effect of peroxisome proliferator-activated receptor δ (PPARδ) agonist GW501516 on the apoptosis induced by oxidized low-density lipoprotein(ox-LDL) or high concentration of glucose in human umbilical vein endothelial cells (HUVECs). METHODS: Cell apoptosis was induced by ox-LDL or high concentration of glucose in HUVECs and was examined by flow cytometry.The HUVECs were treated with GW501516 at different concentrations. The viability of HUVECs was analyzed by MTT assay. RESULTS: The apoptosis rate of HUVECs treated with ox-LDL was 21.3%, while those of HUVECs treated with ox-LDL combined with low, medium and high concentrations of GW501516 were 17.47%, 9.72% and 3.94%, respectively. The apoptosis rate of HUVECs treated with glucose was 22.60%, while those of HUVECs treated with glucose combined with different concentrations of GW501516 were 20.23%, 17.01% and 9.38%, respectively.The results indicated that ox-LDL or glucose induced apoptosis of HUVECs and GW501516 decreased the apoptotic rates induced by ox-LDL or glucose in a dose-dependent manner. The results of MTT assay showed that glucose or ox-LDL decreased the viability of HUVECs and GW501516 attenuated the effect of glucose or ox-LDL on HUVECs. CONCLUSION: GW501516 inhibits the apoptotic effects of ox-LDL and glucose on HUVECs and increases the viability of the cells.     

Effects of high glucose on proliferation,migration, adhesion and secretion of rat late endothelial progenitor cells

AIM: To investigate the effects of high glucose on the proliferation, adhesion, migration and secretion potentials of late endothelial progenitor cells (EPCs) from bone marrow. METHODS: Mononuclear cells were collected from rat bone marrow by density gradient centrifugation and cultured with M199 medium. The early EPCs were identified by DiI-ac-LDL and FITC-UEA-1 double staining. The late EPCs were identified by RT-PCR to detect the expression of von Willebrand factor (vWF) and VE-cadherin. Moreover, the cells were identified by FACS to detect the expression of CD133 and vascular endothelial growth factor receptor-2(VEGFR-2). The 3rd generation of EPCs was harvested and incubated with glucose in a series of concentrations (5, 10, 20 or 40 mmol/L). The cell proliferation, adhesion, migration and the secretion of chemokines such as monocyte chemoattractant protein-1(MCP-1) and interleukin-8 (IL-8) were assayed with MTT, adhesion test, modified Boyden chamber assay and ELISA, respectively. RESULTS: Compared with normal glucose (5 mmol/L)treatment, high glucose (10, 20, 40 mmol/L) dose-dependently degraded the proliferation and migration of late EPCs (P<0.05 or P<0.01). At the same time, treatment with glucose at the concentration of 40 mmol/L decreased the adhesion of EPCs (P<0.05) and increased the release of MCP-1 and IL-8 by late EPCs. CONCLUSION: High glucose inhibits proliferation, adhesion and migration of late EPCs, and enhances the secretion of inflammatory factors, indicating that the high glucose correlates with the vascular complications of patients with diabetes.     

小鼠小肠上皮细胞的分离培养研究

通过组织块培养法进行胎鼠原代细胞培养,采用刮除法和相差消化及相差贴壁法对细胞进行纯化,用0.05%的胰蛋白酶进行消化传代对小鼠小肠上皮细胞进行了原代分离培养、传代、纯化及鉴定。结果表明:组织块培养法得到了小鼠生长状况良好的细胞,经过纯化,得到了较纯的小肠上皮细胞;形态学观察和免疫组化检测显示得到的细胞为小鼠上皮细胞。用组织块培养可以分离纯化出增殖能力较强的上皮细胞,并能进行传代纯化,通过免疫组化鉴定,证明分离出的小鼠IEC为阳性,即为小肠上皮细胞。     

四氯联苯对鸡胚生殖新月原始生殖细胞分布的影响

为了探索四氯联苯(2,2′,5,5′)对鸡胚生殖新月原始生殖细胞(PGCs)分布的影响,本试验将四氯联苯注入种蛋的胚盘(第X期)处,在38℃,相对湿度60%,每2h45度转蛋的条件下孵化至原条期,通过不同方法检测原条期生殖新月明区、暗区及血液循环中的PGCs,探讨四氯联苯对胚盘生殖新月区PGCs分布的影响。结果表明,四氯联苯明显降低了原条期明区和血液中PGSs的数量(P<0.01),但对暗区PGCs的数量影响不明显。这说明四氯联苯对血液中PGCs的影响是由于降低了生殖新月明区PGCs的缘故,和暗区的PGCs无相关性。     

猪前体脂肪细胞与肌卫星细胞体外联合培养

利用差速贴壁法分别纯化前体脂肪细胞与肌卫星细胞,按1∶10的浓度比接种混合培养。以单独两类细胞培养作对照,采用油红O染色和Desmin免疫组化染色鉴定,通过形态学观察和MTT比色绘制生长曲线研究细胞生长规律。结果表明,Desmin免疫组化染色观察肌细胞阳性率达97%以上,油红O染色计数诱导分化细胞比率大于60%;形态学观察发现,联合培养细胞接触汇合前形态呈梭形,接触后不规则重叠交织生长;充脂细胞呈现较晚,且数量稀少;HE染色可见多核细胞融合成肌管样结构;测定2～9d细胞生长曲线显示,联合细胞组增殖比率大于对照组,经历2～3d潜伏期、3～7d指数生长期后,并未与对照组同步进入平台期,而呈持续缓慢上升趋势。实验初步实现了体外猪前体脂肪细胞与肌卫星细胞的直接联合培养,并显示其与对照组生长特性存在差异。     

Inhibitory effect of geinistein on apoptosis in hUVECs induced by MCP-1

AIM: To study the inhibitory effect of genistein on apoptosis in human umbilical vein endothelial cells (hUVECs) induced by monocyte chemotactic protein-1 (MCP-1). METHODS: The hUVECs were cultured in vitro and identified. Growth-arrested hUVECs were stimulated with genistein at different concentrations (0.1 μmol, 1.0 μmol, 10 μmol, 100 μmol) and co-treated with MCP-1 (10 μg/L). The survival rates of hUVECs were detected by MTT assay. The cell cycle and DNA content were detected by flow cytometry. To explore the possible mechanism of the genistein interventions, the expressions of Bcl-2, Fas and Bax proteins were detected by flow cytometry and Western blotting.RESULTS: Genistein increased the survival rate and the level of Bcl-2, inhibited Fas and Bax, decreased the ratios of apoptosis compared with MCP-1-induced hUVECs apoptotic group in a dose-dependent-manner. CONCLUSION: Genistein inhibits the apoptosis induced by MCP-1 and the inhibitory effect was relative to the dose of genistein. Its mechanism might be involved in the down-regulation of Fas and Bax expressions and the up-regulation of Bcl-2.