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目的探讨人主功脉-性腺-中肾(AGM)区基质细胞对脐血 CD34~+细胞的造血支持作用。方法采用免疫磁珠法分离人脐血 CD34~+细胞,接种于底层已制备好的人 AGM 区基质细胞 S1～S5饲养层的24孔板中,同时设无饲养层组及取自同期胚胎的躯干成纤维(hFT)细胞为对照,体外培养28 d,每7 d 收获细胞并检测总数,流式细胞术检测 CD34~+、CD34~+ CD38~-细胞含量,并行造血细胞集落培养。结果在未加外源性造血生长因子的条件下,5株人 AGM 区基质细胞 hAGMS1～S5对制造血细胞总数、CD34~+及 CD34~+CD38~-细胞、造血细胞集落均有不同程度的扩增及维持作用,与无饲养层组、hFT 细胞组比较差异有统计学意义(P<0.05)。细胞总数在21d 达到峰值[扩增(25.13±4.83)倍],CD34~+、CD34~+CD38~-细胞在扩增14 d 达到峰值[(2.68±0.51)倍、(2.38±0.45)倍],高增殖潜能集落形成单位(HPP-CFU)亦在14 d 达到峰值[(2.62±0.85)倍]。hAGMS1～S5的造血支持作用组间比较差异亦有统计学意义(P<0.05),其中 hAGMS3、S4优于 S1、S2及 S5。结论人 AGM 区基质细胞对脐血 CD34~+细胞具有维持及扩增作用,特别是 hAGMS3、S4两株细胞具有更好的维持作用,为胚胎早期造血发生机制和诱导胚胎干细胞造血分化研究提供了重要的模式细胞和基础资料
Objective To investigate the hematopoietic progenitor effect of stromal cells of human main gonad-gonadal-mesonephros (AGM) on cord blood CD34 ~ + cells. Methods Human umbilical cord blood CD34 ~ + cells were isolated by immunomagnetic beads method and inoculated into 24-well plates of well-prepared human AGM stromal cells S1 ~ S5 feeder layer. At the same time, feeder-free group and embryos Trichoderma fibroblasts (hFT) cells were used as control and cultured in vitro for 28 days. Cells were harvested every 7 days and assayed. Flow cytometry was used to detect the percentage of CD34 +, CD34 + CD38 ~ -cells and parallel hematopoietic cell colony culture. Results In the absence of exogenous hematopoietic growth factor, hAGMS1 ~ S5 of 5 human AGM stromal cells had different degrees of expansion on the total numbers of blood cells, CD34 ~ +, CD34 ~ + CD38 ~ - cells and hematopoietic colonies Compared with the feeder-free group and hFT group, the difference was statistically significant (P <0.05). The total number of cells peaked at 21 days [(25.13 ± 4.83) fold]. The peak of CD34 ~ + and CD34 ~ + CD38 ~ - cells peaked at 14 days (2.68 ± 0.51 times, 2.38 ± 0.45 times) (HPP-CFU) peaked at 14 days [(2.62 ± 0.85) fold]. The hAGMS1 ~ S5 hematopoietic supporting effect was also statistically significant difference between groups (P <0.05), hAGMS3, S4 is superior to S1, S2 and S5. CONCLUSIONS: Human AGM stromal cells maintain and amplify cord blood CD34 ~ + cells, especially hAGMS3 and S4 cells. These two cells have a better maintenance effect on embryonic hematopoiesis and induction of hematopoietic differentiation of embryonic stem cells Important model cells and basic information