重组人血小板生成素对体外培养基质细胞的影响

本研究观察rhTPO有无促进骨髓纤维化的作用。用改良Dexter培养法进行体外不同浓度rhTPO作用下的基质细胞培养，在培养的不同时间用MTT法检测细胞增殖活性，用光学显微镜和扫描电子显微镜观察细胞形态变化，用细胞化学方法观察细胞ALP、PAS、AS—DNCE及纤维染色的变化，用免疫组织化学方法检测纤维连接蛋白，及层黏蛋白和Ⅳ型胶原的变化，用流式细胞术检测细胞表面抗原。结果表明：rhTPO可刺激基质细胞增殖，且随浓度增加而增高；细胞增殖活性随浓度增加而增强，但不随作用时间延长而增加；培养第3天，各组细胞明显贴壁，细胞呈椭圆形，第7天可见散在分布的梭形细胞，第12—14天，细胞排列呈一定的方向性，似漩涡状，细胞为长梭形，70％-80％的瓶底被细胞覆盖。第16—18天细胞覆盖达90％以上，细胞形态均为螺旋状排列的长梭形，类似成纤维细胞形态，瑞氏-姬姆萨染色显微镜下观察主要为成纤维细胞，还有极小部分巨噬细胞，内皮细胞和脂肪细胞，对照组与实验组之间无明显差异。培养14—42天的贴壁细胞，PAS显示为阳性，ALP染色和氯醋酸AS—D萘酚酯酶染色显示为弱阳性，对照组与实验组之间无明显差异；用Masson氏三色和Gomori染色法染色，胶原纤维和网状纤维显示均为阴性；纤维连接蛋白，层黏蛋白和Ⅳ型胶原染色在对照组与实验纽均显示为阳性，但实验组阳性均似稍强，这种阳性并没有随着培养时间的延长而增强；在扫描电镜下观察到随着培养时间延长，细胞表面的绒毛、纤维从无到有，细胞从单层到多层，并逐渐出现新生成的纤维细胞，但对照组与实验各组之间未见明显差异；rhTPO对CD34、CD45、CD105、CD106、CD166的表达无明显影响。结论：rhTPO不影响基质细胞的组织化学特性，纤维染色和扫描电镜观察也未发现有促进纤?

Effects of Recombinant Human Thrombopoietin on Stromal Cells in Culture in Vitro

This study was aimed to investigate whether the thrombopoietin （rhTPO） may facilitate myelofibrosis or not. The modified Dexter culture system with various concentrations of rhTPO was used to culture the stromal cells in vitro; the proliferative activity of cells was detected by MTF method; the morphologic changes were observed by light and scanning electron microscopy; the staining changes of ALP, PAS, AS-D NCE and Ⅳ type collagen were observed by cytochemistry method; the changes of fibronectin, laminin and Ⅳ type collagen were assayed by immunohistochemistry method; the cell surface antigens were assayed by flow cytometry. The results indicated that rhTPO could promote the proliferation of stromal cells which was related to the concentrations of rhTPO. Proliferative activity of stromal cells increased with increasing of rhTPO concentration, and was not related to the exposure time. On day 3 stromal cells adhered to the wall, and became oval. On day 7 stromal cells turned to fusiform and scattered dispersively. On day 12 to 14 these cells ranged cyclically and became long fusiform. Cells covered 70% - 80% area of bottle bottom at that time. By day 16 to 18 these cells covered more than 90% area of bottom and ranged cyclically. They displayed the same shape as fibroblasts. By light microscopy with Wrights-Giemsa staining, fibroblasts predominated morphologically, few macrophages, endothelial cells and adipose cells were found. There were no significant differences between experimental group and control group. On day 14 to 42 the adherent cells were positive with PAS staining, poorly positive with ALP and naphythol AS-D chloroacetate esterase （AS-D NCE ） staining, and the difference in cytochemistry was not significant between two groups. When these cells were dyed with Masson＇s trichrome and Gomori＇s staining, neithercollagen fibers nor reticular fibers were positive, but fibronectin, laminin, and collagen type Ⅳ appeared positive stronger in experimental group than those in control. The expres