不同相对分子质量透明质酸对兔骨髓来源间充质干细胞成软骨分化的影响

The effects of exogenous hyalnronic acid with different molecular weight on ehondrogenic differentiation of rabbit bone marrow mesenchymai stem cells

目的观察不同相对分子质最透明质酸（HA）对兔骨髓来源间充质干细胞（MSCs）向软骨方向定向分化的影响。方法取P3代术诱导的骨髓来源间充质干细胞，培养液中分别添加不同相对分子质量透明质酸做诱导剂，分为3个实验组，A组：相对低分子质量组（基础培养基＋HA0．1g/L，相对分子质疑约1000×10^3），B组：相对中分子质量组（基础培养基＋HA0．1g／L，相对分子质量约1800×10^3），C组：相对高分子质量组（基础培养基＋HA0．1g/L，相对分子质量约2000×10^3）。同时设立阳性对照组[基础培养基＋10μg／L转化生长因子-β3（TGF-β3）]及空白对照组（基础培养基），观察细胞形态及增殖情况，分别于诱导后第7、14、21天行甲苯胺蓝染色检测蛋白聚糖表达，另外采用免疫组织化学染色及逆转录-聚合酶链反应（RT—PCR）方法检测细胞Ⅱ型胶原表达。结果经添加软骨诱导剂后，干细胞细胞增殖速度放缓，形态逐渐改变，细胞外基质呈甲苯胺蓝异染性，Ⅱ型胶原免疫组织化学染色阳性。RT—PCR检测示实验组Ⅱ犁胶原mRNA表达阳性，诱导至21d可见各实验组Ⅱ弄胶原基因相对表达量分别为0．64±0．06、0．72±0．03、0．75±0．01，与阴性对照组（0．09±0．03）、阳性对照组（0．96±0．15）比较差异均有统计学意义，但B、C组间Ⅱ型胶原表达相似。结论不同相对分子质量外源件HA诱导兔MSCs向软骨细胞分化的能力存在差异，相对高分子质量的HA的诱导能力较相对低分子质量HA的诱导能力强。证明透明质酸的相对分子质量与MSCs的软骨分化有关联性，但均比TGF—β3的诱导能力弱。

Objective To study the effects of exogenous hyaluronic acid （HA） with different molecular weight on ehondrogenic differentiation of rabbit bone marrow-derived mesenchymal stem cells （MSCs）. Methods The un-induced MSCs in passage 3 were divided into five groups with different suplcments： Group A, basic medium ＋HA 0. 1 g/L （average molecular weight： 1000 ×10^3）; Group B, basic medimn ＋ HA 0. 1 g/L （ average molecular weight 1800×10^3 ） ; group C, basic medium ＋ HA 0. 1 g/L （ average molecular weight 2000 × l0^3 ） ; Group D, basic medium ＋ transforming growth factor-β3 （ TGF-β3） 10μg/L （positive control） ; Group E, basic medium only （negative control）. The morphologic features and proliferation of MSCs were observed under a light microscope. The chondrogenie differentiation was identified by toluidine Hue staining as well as immunohistochemistry and reverse transcription-polymer- ase chain reaction （ RT-PCR ） were used to detect the expression of collagen l/ respectively. Results The speed of cell proliferation was decelerated ＂after HA was added. The cell morphology changed from long spindle to polygon, oval. The extracellular matrix showed metachromasia with toluidine blue and positive type Ⅱ collagen imnmnohistoehemical staining. The mRNA expression of type lI collagen was detected by RT-PCft. The relative gone expression of type II collagen in group A, B and C was 0. 64± 0. 06, 0. 72 ±0. 03, 0. 75±0. 01, respectively, and that in groups D and E was 0. 96 ＋ 0. 15 and 0. 09 ＋_ 0. 03 respectively. There was uo statistically statistically difference in the expression of type Ⅱ collagen between groups B and group C. There was significant differenee between experimental group and control groups. Conclusion The exogenous HA with different molecular weight can induce chondrogenie differentiation of MSCs with different ability. HA with higher average molecular weight enhances the chondrogenesis of MSCs effectively. But all HA shows weaker ability of inducement than TGF-β3.