髌下脂肪垫源间充质干细胞的无酶法获取及修复兔膝关节软骨病损的研究

Study on non-enzymatic extraction of infrapatellar fat pad mesenchymal stem cells and efficacy of rabbit knee cartilage lesions repair

目的探究髌下脂肪垫源间充质干细胞(infrapatellar fat pad mesenchymal stem cells,IPFP-MSCs)的分离及对膝关节软骨损伤的修复效果。方法以雄性新西兰大白兔为实验动物,随机选择其中6只,分别使用无酶法与酶消化法分离提取IPFP-MSCs,检测其形态特征与免疫表型。爬片法与成软骨小球法验证其分化潜能,并通过PCR检测成软骨分化标志物COL2、SOX9、ACAN、COL10。随后,将剩余24只实验兔随机分为4组,建立膝关节软骨损伤模型,并分别使用无酶法分离的IPFP-MSCs(A组)、酶消化法分离的IPFP-MSCs(B组)、无酶法分离的基质血管成分(stromal vascular fraction,SVF)(C组)以及生理盐水(D组)进行关节腔注射,随后监测各组软骨修复变化。结果光镜检查结果显示,使用无酶法提取的IPFP-MSCs与酶消化法提取的IPFP-MSCs具有较为一致的形态特征,流式检测结果显示出具有CD44、CD90阳性,CD34、CD45阴性的特征。细胞活性的检测结果也显示,通过无酶法提取的IPFP-MSCs虽然集落形成率要略低于酶消化法提取的IPFP-MSCs,但其细胞形态与生长状态差异无统计学意义。此外,无酶法分离的IPFP-MSCs中COL2、SOX9、ACAN分别为(1.02±0.09)、(1.04±0.24)、(1.01±0.30),与酶消化分离的IPFP-MSCs中COL2、SOX9、ACAN表达水平(0.97±0.12)、(0.92±0.16)、(1.21±0.18)比较同样差异均有统计学意义(P>0.05)。而动物实验结果显示,A、B组实验兔的软骨病损区域内均有明显新生的组织填充,可生成较为连续的新生软骨层。结论通过无酶法分离提取的IPFP-MSCs具有典型的脂肪间充质干细胞形态特征,对于膝关节软骨损伤兔的软骨组织有良好的改善效果,相比于有酶法,无酶法具有更高的经济效应,有望成为未来临床治疗膝关节软骨损伤的新方案。

Objective To evaluate the non-enzymatic isolation method of infrapatellar fat pad mesenchymal stem cells(IPFP-MSCs)and the efficacy in treating knee cartilage lesions in rabbits compared with traditional enzymatic isolation method.Methods Mature male New Zealand white rabbits were used as experimental animals.IPFP-MSCs were isolated by non-enzymatic and enzymatic method respectively from 6 of the rabbits randomly selected.IPFP-MSCs were characterized via flow cytometry as well as their potential for differentiation into adipocytes,osteoblasts,and chondrocytes.The markers of chondroblast differentiation COL2,SOX9,ACAN and COL10 were tested by RT-PCR.Then,the remaining 24 experimental rabbits were randomly divided into 4 groups,and knee joint cartilage lesions were established in each group by surgery.IPFP-MSCs isolated by non-enzymatic method(group A),IPFP-MSCs isolated by enzymatic method(group B),SVF isolated by non-enzymatic method(group C)and saline(group D)were injected into articular cavity respectively.Distal femur samples were evaluated at 6-and 12-weeks posttreatment via macroscopic observation and histological assessment based on the International Cartilage Repair Society(ICRS)visual histological assessment scale.Results The morphological characteristics of IPFP-MSCs isolated by enzymatic and non-enzymatic method were similar observed by light microscopy.Flow cytometry showed that IPFP-MSCs were CD44+CD90+CD34-CD45-.The colony formation rate of IPFP-MSCs extracted by non-enzymatic method was slightly lower than that of IPFP-MSCs isolated by enzymatic method.No significant differences were found in cell morphology and growth state.In addition,COL2,SOX9,and ACAN in IPFP-MSCs isolated via non-enzymatic method were(1.02±0.09),(1.04±0.24),and(1.01±0.30),respectively;the expression levels of COL2,SOX9,and ACAN in IPFP-MSCs isolated by enzymatic method were(0.97±0.12),(0.92±0.16),and(1.21±0.18);no significant differences were noted between the two groups(P>0.05).The results of animal experiments showed that there was obvious new tissue filling in the lesion area of experimental rabbits in group A and B,with relatively continuous new cartilage layer formed.Conclusions IPFP-MSCs isolated by non-enzymatic method have typical morphological characteristics of adipose mesenchymal stem cells and excellent improvement effect on cartilage tissue of rabbits with knee cartilage injury.Compared with enzymatic method,non-enzymatic method has more clinical transformation value,and is expected to become a new clinical treatment for knee cartilage injury in the future.