差速贴壁及克隆化培养人胚成肌细胞及其移植恒河猴的实验性研究

Differential Attachment and Cloning Culture of Human Fetal Muscle Myoblast and Its Experimental Transplantation in Fujian Rhesus Monkeys

６个水囊引产４～５个月龄人胚肢体肌组织，采用０．１％Ⅱ型胶原酶和０．１２５％胰蛋白酶二步消化法，分离肌卫星细胞，在含胎肌条件培养液（ＨＦＭＥ）的培养基中，差速贴壁及克隆纯化，电镜及Ｄｅｓｍｉｎ免疫细胞化学鉴定纯度后，Ｒｕｂｂｅｒｐｏｌｉｃｅ刮除收集，按１．６×１０６个细胞／ｋｇ体重，间隔０．５ｃｍ点均匀移植于恒河猴肱二头肌组织中。结果表明，该方法培养的成肌细胞纯度达９６％以上，移植后血清肌浆酶ＬＤＨ及ＣＰＫ与移植前相近（Ｐ＞０．０５），肌组织活检未见明显坏死，表明成肌细胞移植无宿主肌纤维损伤作用；移植后１周ＣＤ４／ＣＤ８比值大于２，补体Ｃ４短暂下降，但Ｔａｃ各值未超过移植前，２周后ＣＤ４／ＣＤ８比值恢复到正常水平，表明恒河猴对移植人的成肌细胞有排异反应，但可以耐受。移植后１周肌组织中可以检出Ｄｅｓｍｉｎ（＋）成肌细胞生长及移植后２周的ＡＯ（＋）的嗜碱性再生肌纤维，此后早期呈ＨＬＡ－ＤＲ（＋）的ＣＤ３（＋）、ＣＤ８（＋）细胞几尽消退。

This paper reported a modified myoblasts culture tech11iqtie. After fi11ely minced fragments of six fresh human fetal muscles were enzymatically dispersed by 0.1%collegenase type Ⅱ-RPMI 1640 and 0.125% trypsin-0.05% EDTA-D-Hanks,the myoblasts were cultured by differential attachment and cloned to purify in the media containing HFME estab lished previously.The cultured myoblasts were lso identified by electron microscopy and desmin immunocytochemistry.The results showed that this method could obtain highly purified myoblasts with purity above 96%.After being gathered,the myoblasts were transplanted in biceps muscle of six Fujian Rhesus monkeys by 0.5 cm interval injections. with 1.6×106 cells/kg bodyweight.The post-MT serum sarcoplasm enzymes,immune reaction and histopathological changes were of injected sides dynamically observed. The results show that this injection transfer was safe, LDH and CPK were not changed significantly(P>0.05),and no severe necrotic change was found.Although the CD4/CD8 ratio was over 2 in one week post-MT,it recovered to normal level after two weeks post-MT. These indicated that immune rejection could happen,butit was slight.One week post-MT,myoblasts survived well among injected myofibers.After two weeks post-MT,regenerated myofibers (desmin and AO-RNA positive)were scattered in the injected muscle and HLA-DR positive T and CTLcells almost disappeared.