CAJ | 학술논문

目的观察不同相对分子质量透明质酸(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代未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+ HA 0.1 g/L,相对分子质量约1000×103),B组:相对中分子质量组(基础培养基+HA0.1 g/L,相对分子质量约1800×103),C组:相对高分子质量组(基础培养基+HA 0.1 g/L,相对分子质量约2000×103).同时设立阳性对照组[基础培养基+ 10 μg/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型胶原mRNA表达阳性,诱导至21 d可见各实验组Ⅱ型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性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.
목적 관찰불동상대분자질량투명질산(HA)대토골수래원간충질간세포(MSCs)향연골방향정향분화적영향.방법 취P3대미유도적골수래원간충질간세포,배양액중분별첨가불동상대분자질량투명질산주유도제,분위3개실험조,A조:상대저분자질량조(기출배양기+ HA 0.1 g/L,상대분자질량약1000×103),B조:상대중분자질량조(기출배양기+HA0.1 g/L,상대분자질량약1800×103),C조:상대고분자질량조(기출배양기+HA 0.1 g/L,상대분자질량약2000×103).동시설립양성대조조[기출배양기+ 10 μg/L전화생장인자-β3( TGF-β3)]급공백대조조(기출배양기),관찰세포형태급증식정황,분별우유도후제7、14、21천행갑분알람염색검측단백취당표체,령외채용면역조직화학염색급역전록-취합매련반응(RT-PCR)방법검측세포Ⅱ형효원표체.결과 경첨가연골유도제후,간세포세포증식속도방완,형태축점개변,세포외기질정갑분알람이염성,Ⅱ형효원면역조직화학염색양성.RT-PCR검측시실험조Ⅱ형효원mRNA표체양성,유도지21 d가견각실험조Ⅱ형효원기인상대표체량분별위0.64±0.06、0.72±0.03、0.75±0.01,여음성대조조(0.09±0.03)、양성대조조(0.96±0.15)비교차이균유통계학의의,단B、C조간Ⅱ형효원표체상사.결론 불동상대분자질량외원성HA유도토MSCs향연골세포분화적능력존재차이,상대고분자질량적HA적유도능력교상대저분자질량HA적유도능력강.증명투명질산적상대분자질량여MSCs적연골분화유관련성,단균비TGF-β3적유도능력약.
Objective To study the effects of exogenous hyaluronic acid (HA) with different molecular weight on chondrogenic 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 suplements:Group A,basic medium + HA 0.1 g/L (average molecular weight:1000 × 103 ) ; Group B,basic medium + HA 0.1 g/L ( average molecular weight 1800 × 103 ) ; group C,basic medium + HA 0.1 g/L ( average molecular weight 2000 × 103 ) ; Group D,basic medium + transforming growth factor-33 ( TGFβ3) 10 μg/L (positive control) ; Group E,basic medium only (negative control).The morphologic features and proliferation of MSCs were observed under a light microscope.The chondrogenic differentiation was identified by toluidine blue staining as well as immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) were used to detect the expression of collagen Ⅱ 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 immunohistochemical staining.The mRNA expression of type Ⅱ collagen was detected by RT-PCR.The relative gene 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 no statistically significant difference in the expression of type Ⅱ collagen between groups B and group C.There was significant difference between experimental group and control groups.Conclusion The exogenous HA with different molecular weight can induce chondrogenic 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-33.