益气活血方含药血清对大鼠骨髓间充质干细胞体外增殖的影响

目的：探讨益气与活血中药含药血清对骨髓间充质干细胞（ bone marrow mesenchymal stem cells,BMSCs ）增殖能力的影响。方法全细胞贴壁培养法获取、纯化BMSCs,体外培养,建立大鼠骨髓间充质干细胞的培养体系,采集空白对照大鼠、益气药、活血药、益气活血中药灌胃大鼠含药血清,用10%浓度以上不同含药血清培养第三代BMSCs,置于37℃,5% CO2培养箱中孵育。用流式细胞术鉴定BMSCs。噻唑基四唑比色法（ methyl thiazolyl tetrazolium,MTT）测细胞活性,绘制生长曲线,观察10%益气、活血、益气活血中药含药血清对大鼠骨髓间充质干细胞体外增殖的影响。结果采用全骨髓贴壁法获取的BMSCs,经流式细胞术检测,CD44、CD106呈阳性表达,基本不表达CD45、CD34；MTT法检测结果显示,培养第2、4、5天细胞快速增长,均较前一天增长明显（ P 〈0．05）,第6天时活血方血清组与益气活血方血清组细胞增长已呈下降趋势,而其它三组在第6天细胞增长达最大,与前一天比较仍有差异（ P〈0．05）,到第7天呈下降趋势。 BMSCs去除基线增殖率组间比较,培养第2天时,各组间比较虽无差异,但活血方血清组达最大（71．39±177;4．98%）；第4、6天时,益气活血方组达最大（66．40±177;1．47%）,与其它三组比较P〈0．01,活血方血清组次之；培养第6天时,益气方血清组较其它三组大,益气活血方血清组次之,活血方血清组最低。结论全细胞贴壁培养的大鼠骨髓间充质干细胞在细胞形态与表形表达上均具备BMSCs特征；益气活血中药含药血清对培养BMSCs增殖影响最大,活血方含药血清对细胞增殖影响出现最早、益气方含药血清对细胞增殖影响时间最长。

Effect of qi-tonifying and blood-activating Chinese herb serum on proliferation of SD rat bone mar-row mesenchymal stem cells in vitro

Objective To explore the qi-tonifying and blood-activating Chinese herb serum on pro-liferation of SD rat bone marrow mesenchymal stem cells （ BMSCs） in vitro. Methods Rat BMSCs were i-solated from rat bone marrow and expanded by whole bone marrow adherent culture method in vitro, collect-ed blank serum of rat as control group and serum from qi-tonifying Chinese herb, blood-activating Chinese herb, qi-tonifying ＆amp; blood-activating Chinese herb gavaged rats. BMSCs of the third generation were cul-tured in vitro with 10% concentrations of these four kinds of medicated serum at 37 ℃ and 5% CO2 incu-bator respectively. Flow cytometry was used for the identification of BMSCs and the proliferation of BMSCs was observed by MTT method and drew the growth curve. Results The BMSCs were obtained by whole bone marrow adherent culture method. The membranes of third generation of BMSCs were positive for CD44 and CD106 and negative for CD45 and CD34 detected by flow cytometry. The results showed that BMSCs grown rapidly compared to the day before it selves to the 2nd, 4th, and 5th days, the difference was signifi-cantly （ P〈0. 05 ） . The cell growth of the blood-activating Chinese herb serum group and qi-tonifying ＆amp;blood-activating Chinese herb serum group showed a downward trend, while the cell growth of the other three groups reached peak on the 6th day, but still had differences compared to the 5th day （P〈0. 05）, and declined on the 7th day. The removed baseline proliferation rate of BMSCs had no differences comparison a-mong groups at 2nd day, but the removed baseline proliferation rate of BMSCs in blood-activating Chinese herb serum group reached maximum （71. 39 ＆#177; 4. 98%）, but no differences compared with the other three groups （P＆gt;0. 05）. Compared with the other three groups, the removed baseline proliferation rate of qi-tonifying ＆amp; blood-activating Chinese herb serum group was the largest （ 66. 40 ±177; 1. 47%） （ P〈0. 01 ） on the 4th and 6th days, followed by blood-activating Chinese herb serum group. The removed baseline prolifer-ation rate of qi-tonifying Chinese herb serum group was the highest on the 6th day, followed by qi-tonifying＆amp;blood-activating Chinese herb serum group and blood-activating Chinese herb serum group. Conclusion BMSCs of SD rat isolation and cultivation by whole bone marrow adherent culture method could steadily express the bone mesenchymal stem cells surface markers and cells morphology. Qi-tonifying＆amp;blood-acti-vating Chinese herb serum has the greatest effect on the proliferation of cultured BMSCs; blood-activating Chinese herb serum motivates cell proliferation earliest; qi-tonifying Chinese herb serum has the longest effects on cell proliferation.