重组人结缔组织生长因子对大鼠脂肪源性干细胞Ⅰ型胶原蛋白合成及分化的影响

Influence of recombinant hCTGF on collagen type I secreting function and differentiation of rat adipose tissuederived stem cells

目的探讨重组人结缔组织生长因子(hCTGF)对大鼠脂肪源性干细胞(ADSCs)分化及Ⅰ型胶原蛋白表达的影响。方法 2010年9月至2011年9月在广州医学院第一附属医院采用贴壁培养法分离纯化大鼠ADSCs,流式细胞仪分析第3代大鼠ADSCs表面抗原标记物;含100μg/L重组hCTGF的培养基作为刺激组,无重组hCTGF为对照组,分别培养7、14、28d后,RT-PCR和western-blot检测大鼠ADSCs中Ⅰ型胶原蛋白表达;观察重组hCTGF对大鼠ADSCs成骨、成脂方向分化的影响。结果大鼠ADSCs表面抗原CD44、CD90、CD105、CD49d阳性,CD34、CD45、CD106阴性;Ⅰ型胶原蛋白mRNA表达量刺激组与对照组7、14、28d时分别为1.069±0.324vs.0.298±0.029、2.162±0.038vs.0.652±0.024、3.360±0.058vs.0.815±0.041,差异有统计学意义(P<0.05),Ⅰ型胶原蛋白的表达量7、14、28d时分别为0.761±0.041vs.0.679±0.029、0.834±0.115vs.0.708±0.061、0.774±0.180vs.0.720±0.026,差异有统计学意义(P<0.05)。结论重组hCTGF可上调大鼠ADSCsⅠ型胶原蛋白的表达,抑制大鼠ADSCs向成脂、成骨分化。推测重组hCTGF可能使大鼠ADSCs发生了向成纤维细胞定向分化。

Objective To investigate the effects of recombinant hCTGF on the proliferation and differentiation of rat ADSCs, and to observe the changes in expression of collagen Ⅰ . Methods In the First Affiliate Hospital of Guangzhou Medical University between September 2010 and September 2011, ADSCs were separated and purified from rat inguinal fat pads using attachment culture method. Surface antigen of the third-generated rat ADSCs was determined by using flow cytometry. Subsequently, the third-generated rat ADSCs were induced with recombinant hCTGF special inducing system in 1- 4week culture, the expression of collagen Ⅰ were measured by RT-PCR and Western blot. In addition, the rat ADSCs which were stimulated by 100μg/L recombinant hCTGF for 14 days were treated by osteogenic and adipogenic medium, to identify the changes of its differentiation potential of osteogenic and adipogenic. Results Rat ADSCs were separated and purified using attachment culture method. The third-generated rat ADSCs were positive for surface antigen CD44, CD90, CD105, CD49d,but negative for surface antigen CD34, CD45 and CDI06. Expression of Collagen Ⅰ mRNA in rat ADSCs were increased with the recombinant hCTGF treatment for 7, 14,28 days（ 1.069 ±0. 324vs. 0. 298± 0. 029,2. 162±0. 038vs. 0. 652±0. 024,3. 360 ±0. 058vs. 0. 815 ±0. 041 ,P 〈 0. 05）. Western blot showed that the expression of collagen Ⅰ was upregulated with the recombinant hCTGF treatment （ 0. 761 ± 0. 041vs. 0. 679 ± 0. 029, 0. 834 ± 0. 115vs. 0. 708 ± 0. 061,0. 774 ± 0. 180vs. 0. 720 ± 0. 026, P 〈 0. 05 ）. The stimulated cells which cultured in osteogenic medium did not format extracellular deposition, and alizarin red staining was negative after cultured for 14 days. Conclusion Recombinant hCTGF can promote the expression and secretion of collagen Ⅰ in rat ADSCs, remove the osteogenic and adipogenic differentiation potential of rat ADSCs.