人骨髓间充质干细胞与脐静脉内皮细胞体外共培养的研究

目的观察人骨髓间充质干细胞（Human bone marrow mesenchymal stromal cells,hBMSCs）与脐静脉内皮细胞（Human umbilical vein endothelial cells,hUVECs）体外共培养对hBMSCs成骨作用及hUVECs成血管功能的影响。方法分离鉴定hBMSCs和hUVECs,将hBMSCs及hUVECs按1∶1比例直接共培养,倒置相差显微镜观察细胞的生长特性和细胞相容性,Matrigel实验观察共培养对hUVECs体外血管形成能力的影响。将hBMSCs成骨诱导7 d后,茜素红染色观察共培养对其成骨分化能力的影响。结果 hBMSCs与hUVECs体外共培养细胞相容性良好,Matrigel实验显示共培养组形成的毛细血管状结构较单一细胞组多。成骨诱导的hBMSCs中,共培养组茜素红染色阳性,单一细胞组茜素红染色阴性。结论 hBMSCs与hUVECs具有良好的细胞相容性,在共培养体系中,未诱导的hBMSCs能促进形成毛细血管状结构,已成骨诱导的hBMSCs成骨矿化能力增强。     

MDCK、Vero和Hep-2细胞共培养分离甲型H1N1型流感病毒

目的用MDCK、Vero和Hep-2细胞共培养体系,分离甲型H1N1流感病毒,提高流感监测效率及缩短临床诊断周期。方法选取4株经国家流感中心复核鉴定、本实验室保存的甲型H1N1流感病毒,用病毒培养液1:2稀释后分别接种MDCK、Vero和Hep-2共培养细胞瓶和MDCK细胞瓶,每组设空白对照,用生理盐水代替接种。经35℃、5%CO2培养后记录甲型H1N1型流感病毒毒株在共培养细胞瓶和MDCK细胞瓶中的细胞病变效应(CPE)、血凝滴度和荧光定量PCR反应的CT值。结果培养后,2组接种流感病毒的细胞瓶均出现典型细胞病变,MDCK细胞瓶CPE略强于共培养细胞瓶,分离物-70℃冻融2次后检测血凝滴度,2组接种流感病毒的细胞瓶内分离物,血凝滴度均(1∶32;提取分离物核酸,荧光定量PCR检测,CT值均(20。结论用MD-CK、Vero和Hep-2细胞共培养体系可以分离流感病毒,同时还可分离鉴定其它呼吸道病毒。     

缺氧条件下共培养血管内皮细胞对C6胶质瘤细胞促进生长和抑制凋亡的作用

目的 研究缺氧条件下共培养血管内皮细胞对C6胶质瘤细胞生长及凋亡的影响.方法 采用24孔板Transwell装置共培养C6胶质瘤细胞与人脐静脉内皮细胞,氯化钴模拟细胞缺氧条件.实验分为缺氧细胞共培养组、常氧细胞共培养组、缺氧细胞单独培养组、常氧细胞单独培养组.培养24 h后Annexin V-FITC/PI荧光双染法检测各组C6细胞凋亡情况,流式细胞仪测定细胞凋亡率和细胞周期,连续计数6d各组C6细胞数目并绘制细胞生长曲线. 结果 共培养24 h后,缺氧细胞共培养组C6细胞凋亡率明显低于其他3组,常氧细胞共培养组C6细胞凋亡率低于常氧细胞单独培养组,差异有统计学意义(P＜0.05);与其他3组比较,缺氧细胞共培养组G0/G1期细胞比例明显减少,而S期细胞比例明显增多,与常氧细胞单独培养组比较,常氧细胞共培养组C6细胞G0/G1期比例较少,S期细胞比例较多,差异均有统计学意义(P＜0.05);连续培养6d,各组C6细胞在前2d均进入细胞指数生长期,在2～4 d达到高峰后,形成细胞生长平台期,随后细胞计数下降,进入退化衰亡期.其中缺氧细胞共培养组C6细胞在指数生长期内生长速率最快,生长平台期内细胞峰值数目最多,并较晚进入退化衰亡期. 结论 缺氧和血管内皮细胞可协同作用于C6胶质瘤细胞,减少胶质瘤细胞的凋亡,并加速其生长.     

嗅鞘细胞对骨髓基质干细胞增殖及Galc表达的影响

目的嗅鞘细胞（OEC）和骨髓基质干细胞（BMSC）是两种可供移植用的种子细胞。联合应用两种细胞移植治疗神经疾病是较为乐观的策略,但作用机制尚待研究。方法本实验利用MTT法和RT-PCR技术探讨OEC对体外培养BMSC增殖和基因表达的影响。结果体外培养细胞生长良好,分别经CD44和P75染色鉴定为BMSC和OEC。共培养的BMSC与单独培养的BMSC比较有更强的增殖能力,差异有统计学意义（P〈0.05）。RT-PCR结果显示共培养BMSC中Galc基因表达明显较单纯BMSC者下调,差异有统计学意义（P〈0.05）。本实验还检测了其它多种因子（β-TublinⅢ、APC、TGF-β2、IGF-1、Bcl-2、Bax）表达,但均没有明显变化（P〉0.05）。结论 OEC能促进BMSC增殖,其分子机制可能与OEC调节BMSC表达细胞因子有关,其中Galc是一个值得关注的分子。     

平滑肌细胞对共培养体系滋养细胞侵袭力与MMP-2及MMP-9表达的影响

目的模拟体内环境,建立可保持平滑肌细胞与绒毛外细胞滋养层细胞(EVCT)生物学特性的共培养细胞模型,应用于研究平滑肌细胞与滋养细胞理化特性与滋养细胞的侵袭行为。方法利用组织块培养法培养脐动脉平滑肌细胞,组织块培养、胰酶消化和Percoll梯度沉降,收集纯化人早孕绒毛组织的滋养细胞,免疫组化检测细胞的纯度。将滋养细胞与平滑肌细胞分别放入Transwell的上下小室,观察该模型下滋养细胞形态变化、细胞活力、侵袭力改变与分泌功能等特性。结果免疫组化显示EVCT的细胞角蛋白7阳性表达的细胞数占95%以上,SMC a-actin阳性表达的细胞数也超过95%,证实共培养系统中EVCT和SMC纯度均在95%以上,且生物学特性得以维持。上室中的EVCT保持了其侵袭能力,且平滑肌细胞能促进滋养细胞增殖活性、侵蚀能力及MMP2、MMP9的表达。结论成功地建立了平滑肌细胞与滋养细胞原代共培养系统模型,便于研究滋养细胞侵袭和子宫螺旋动脉重铸障碍的分子机制。     

3T3-L1 adipocytes induce dysfunction of MIN6 insulin-secreting cells via multiple pathways mediated by secretory factors in a co-culture system

Pancreatic β-cell dysfunction is an important pathological change in type 2 diabetes, which is tightly related to obesity. However, the direct role of adipose tissue in β-cell dysfunction has not been well understood. In this study, we examined the effects of 3T3-L1 adipocytes on MIN6 insulin-secreting cells in a co-culture system. MIN6 cells used here kept most of β-cell functions but less sensitive to glucose stimulation. Tolbutamide, the K_ATP channel blocker, was therefore used to stimulate insulin secretion in this report. MIN6 cells co-cultured with 3T3-L1 adipocytes had significantly reduced intracellular calcium concentration ([Ca²⁺]_i) and lost the ability to secrete insulin in response to tolbutamide, compared to the control cells. 3T3-L1 adipocytes significantly decreased the expression of insulin, glucokinase and Kir6.2 genes but increased the expression of uncoupling protein-2 (UCP-2) in MIN6 cells after one week of co-culture, as measured by semi-quantitative RT-PCR. 3T3-L1 adipocyte-conditioned medium also significantly decreased insulin secretion and the expression of insulin, glucokinase and Kir6.2 genes in MIN6 cells. The conditioned medium also reduced tyrosine kinase activity in MIN6 cells. The inhibitor of protein tyrosine kinase, genistein, decreased the expression of glucokinase and Kir6.2 in MIN6 cells, while two free fatty acids, oleic acid and linoleic acids, were found to increase UCP-2 expression. The present study demonstrates that 3T3-L1 adipocytes directly impair insulin secretion and the␣expression of important genes in MIN6 cells. The effects of␣3T3-L1 adipocytes on MIN6 cells are ascribed to␣secreted bioactive factors and may be mediated via multiple pathways, which include the upregulation of UCP-2 expression via free fatty acids, and downregulation of glucokinase and Kir6.2 expression via decreasing protein tyrosine kinase activity.     

Induction of endostatin expression in meniscal fibrochondrocytes by co-culture with endothelial cells

Introduction  The ultimate goal after meniscus damage is the preservation of the original meniscal tissue, which is often impossible due to the limited healing capacity of meniscal lesions, especially in the avascular zone. Factors produced by endothelial cells of meniscal vessels may contribute to better wound healing in vascularized zones. We therefore investigated the expression of different angiogenic factors, growth hormones and cytokines in human fibrochondrocytes and in fibrochondrocytes upon co-culture with endothelial cells, to examine mechanisms of repair of meniscal injury in more detail and to investigate the potential use of endothelial cells in co-cultures for autologous meniscal repair utilizing tissue engineering technology. Materials and methods  Gene expression of SMAD-4, iNOS, IL-1β, VEGF, MMP-1, MMP-3, MMP-13, aggrecan, biglycan, vimentin, collagen-I, -II, -III, -IV, -VI, -X, -XVIII, angiopoietin-1, angiopoietin-2, and thrombostatin-1 were investigated in fibrochondrocytes in comparison to cells in co-culture with human umbilical vein endothelial cells (HUVEC). The expression of endostatin was enumerated in cell supernatants. A proliferation assay was used to investigate the mitotic activity of the cells. Results  In presence of HUVEC, meniscal fibrochondrocytes expressed SMAD-4, iNOS, IL-1β, VEGF, MMP-1, MMP-3, MMP-13, aggrecan, biglycan, vimentin, collagen-I, -II, -III, -VI, and -XVIII at rates comparable to cells without HUVECs. Note that the expression of endostatin was significantly higher in the co-culture when compared to the separate fibrochondrocyte cultures and the proliferation rate of endothelial cells was significantly decreased in co-culture. Conclusion  We conclude that the expression of the anti-angiogenic factor endostatin increased in the fibrochondrocytes. This may limit the regeneration capacities of meniscal injury in vivo.     

大鼠雪旺细胞和成骨细胞联合培养的实验研究

目的观察大鼠雪旺细胞与成骨细胞体外联合培养条件下,成骨细胞的生长和增殖情况。方法日龄3 d的SD大鼠,采用混合酶消化法体外分散培养雪旺细胞和成骨细胞,并用Millicell小室实现雪旺细胞与成骨细胞的联合培养。待细胞生长稳定后,用倒置相差显微镜观察细胞的形态学特征,MTT法检测成骨细胞的增殖情况。结果对照组成骨细胞于10 d左右可以铺满培养板底,此时细胞体积增大、数量增多,形态以多角形为主,细胞之间出现重叠生长,联系密切。共培养组成骨细胞生长速度增快,7 d左右即可铺满培养板底,逐渐形成铺路石状,其中心部位的细胞较密集,可以出现散在的钙化结节。MTT结果显示共培养组成骨细胞增殖明显。结论雪旺细胞对成骨细胞的生长、增殖具有一定的促进作用。但是,关于该作用是否是通过FGFs实现的还有待于进一步的研究。     

Lipopolysaccharide activation of pericyte's Toll-like receptor-4 regulates co-culture permeability

BACKGROUND: Pericytes (PCs) have a synergistic relationship with endothelial cells (MVEC) in regulating capillary permeability. PCs express Toll-like receptor-4 (TLR-4). We hypothesize one mechanism of MVEC/PC co-culture permeability is regulated through lipopolysaccharide (LPS) activation of pericyte TLR-4. METHODS: Rat PCs were harvested and cultured. PCs were transfected with siRNA targeted to TLR-4. Western blotting was used to confirm gene silencing of TLR-4. A previously described co-culture permeability assay was performed after LPS treatment. RESULTS: Western blot confirmed successful silencing of TLR-4 in PCs, which was sustained for 7 days. A dose- and time-dependent effect of LPS on albumin clearance was seen in MVEC/PC co-cultures. Co-cultures with TLR-4 silenced in PCs eliminated the LPS dose-dependent increase in albumin clearance. CONCLUSIONS: TLR-4 regulates pericyte mediated capillary leak seen with LPS exposure. Our TLR-4 silencing model can be used to further investigate TLR-4's role in pericyte mediated capillary leak.