中空纤维灌注培养模拟骨髓造血研究

采用细胞工程技术探索造血细胞体外扩增技术以维持其自我更新潜能，抑制过度分化。方法：首先建立微载体 基质细胞体外造血模型（G1组，即瓶培养模式），设置单纯微载体 基质细胞培养（G2组）和单纯骨髓细胞液体悬浮培养（G3组）作对照。检测各组粒系 巨噬系造血祖细胞集落产率（CFU GM/105）。自行设计1对引物，以检测Balb/c小鼠原始造血细胞c kit基因mRNA表达水平。试用中空纤维模拟血管灌注功能（Gh组，即中空纤维灌注模式），以G1、G2、G3作对照，并对各组培养效果进行评价。结果：微载体 基质细胞体外造血模型实验结果显示：小鼠骨髓细胞培养2周后，CFU GM/105检测G1组比G3组高7.7倍（P<0.05），是2个对照组（G2+G3）集落产率总和的1.9倍。原始造血细胞c kit mRNA表达水平：模型G1组比G2组高3.7倍，比G3组高62.3倍，且差异均显著。在成功建立微载体 基质细胞体外造血模型基础上进行中空纤维灌注培养实验，CFU GM/105检测显示：Gh组比G3组高4.6倍，并且略高于G2组；Gh组与G1组集落产率差别不明显。在原始造血细胞c kit mRNA表达水平上Gh组最高，从Gh、G1、G2到G3依次呈下降趋势。结论：在没有外加细胞因子的条件下，微载体 基质细胞和中空纤维灌注造血模型可抑制造血干、祖细胞过度分化与耗竭，维持其c kit较高的表达水平。

Simulation of Bone Marrow Hematopoiesis Using Perfusion Culture in the Membrane of Hollow Fibers

Objective: To explore a novel technigue to amplify hematopoietic cells, maintaining their self renewal potential and inhibiting their overdue differentiation by using cytotechnology. Methods: At first, a hematopoietic model was established, namely group 1 (G1, also called as “the flask culture system”). In this model, mouse bone marrow cells were cocultured on the stromal cells which covered the growth surface of the microcarriers. At the same time, two control groups, group 2 (G2), the only stromal cells seeded on the microcarriers; and group 3 (G3), the only bone marrow cells, were cultured. The efficiency of colony forming unit of granulocyte macrophage (CFU GM/105) was determined. A pair of oligonucleotide primers was designed to test the expression level of c kit mRNA in primitive hematopoietic cells of Balb/c mouse by RT PCR. Then the membrane of hollow fibers was tried to simulate the function of vascular perfusion, namely group h (Gh, also called as “the hollow fiber perfusion system”). G1,G2 and G3 were cultured simultaneously as control groups. The effects of all the groups on the hematopoietic cells were evaluated after 2 week culture. Results: In the hematopoietic model, after 2 week culture, the efficiency of CFU GM/105 in G1（46.0±27.7） was 7.7 fold higher than that in G3 (5.3±7.8, P<0.05), and was 1.9 fold as high as the sum of G2 and G3. The OD ratio of c kit gene mRNA in the model group G1 (0.759±0.290) was 3.7 folds higher than that in G2(0.163±0.248, P<0.05), and 62.3 folds higher than that in G3 (0.012±0.026, P<0.05). The level of c kit mRNA in G1 was 3.3 folds higher than the sum of two control groups (G2+G3, P<0.05). Initial data demonstrated that hematopoietic model had significant level of c kit mRNA and colony efficiency. Based upon achieving the hematopoietic flask culture system, the perfusion culture was performed in the hollow fiber cartridge. After 2 week culture, the efficiency of CFU GM in the perfusion culture with hollow fiber catridge (Gh, 18.9±20.6) was 4.6 folds higher than that in G3 (3.4±3.7), and a little higher than that in G2 (14.8±25.3) and in the sum of two control groups (G2+G3) . There was not significant difference between Gh and G1, but the colony efficiency in Gh was lower than that in G1. After 2 week culture, the mRNA expression level of c kit gene in Gh was the highest, and showed a descending trend in Gh, G1, G2 and G3 in turn. The perfusion system with the Flame disinfecting Sterile Connector (FSC) using the sealed sterile operation technique was kept in sterile condition for six months. The same designs and results have not been reported yet. Conclusions: Three dimensional culture further simulated the microenvironment of bone marrow hematopoiesis.Without supplemented cytokines, the hematopoietic models of stromal cells adhering onto the microcarries and perfusion with hollow fibers can inhibit hematopoietic stem and progenitor cells from excessively differentiation and exhaustion, and maintain the high level expression of c kit mRNA. The pair of oligonucleotide primers of Balb/c mouse c kit gene designed has many merits such as high specificity and stability. It is feasible that the function of vascular perfusion is simulated with hollow fiber semipermeable membranes. The hollow fiber perfusion system will contribute to further simulate hematopoietic microenvironment, and make it possible to realize ex vivo amplification of hematopoietic cells.