异体角质形成细胞免疫原性的初步研究

RESEARCH OF IMMUNOGENITY OF ALLOGENIC KERATINOCYTES

目的观察组织工程皮肤的种子细胞角质形成细胞体外培养后的免疫原性变化情况。方法取自愿捐献的健康儿童、青少年包皮,采用分离酶、胰蛋白酶两步消化法进行体外培养并传代至5代,行苔盼蓝计数细胞活性。SP法标记各代角质形成细胞中的朗格汉斯细胞和黑色素细胞,计算其占总细胞数的百分比。各代角质形成细胞与从外周血中分离培养淋巴细胞进行混合培养,观察淋巴细胞增殖情况,闪烁仪计数cpm值。结果培养的细胞生长良好,原代及传代后的细胞融合后呈铺路石样生长。细胞冻存后复苏,苔盼蓝染色细胞活力>80%。原代中朗格汉斯细胞占5.8%,黑色素细胞占8.1%;1代中朗格汉斯细胞占2.1%,黑色素细胞占2.8%;2代后二者均消失。cpm值原代482.13±46.61,1代362.50±35.12,2代228.38±51.46,3代171.86±34.63,4代143.63±15.95,5代123.25±14.39,与对照组53.67±8.61比较,差异均有统计学意义(P<0.05)。原代、1代与其他各代比较,2代与4、5代比较,差异均有统计学意义(P<0.05);2代与3代间,3、4代及5代间,差异均无统计学意义(P>0.05)。结论异体角质形成细胞经体外培养后不断传代,细胞免疫原性逐渐下降。

Objective To observe the change of the immunogenity of keratinocytes when cultured in vitro. Methods Health children foreskins were digested by dispase and trypase. The human keratinocytes were cultured in vitro and passaged in succession until the fifth passage. Different passage keratinobytes signed by SP method to show the percentage of langerhans cells and melanocytes. Every passage keratinocytes were respectively mixed with allogenic lymphocytes which isolated from peripheral blood, and then the proliferation degree of allogenic lymphocytes was tested. Results Keratinocytes were cultured well in K-SFM medium. When keratinocytes conjugated, every passage cells grew like paving stone. After cryopreservation and then rewarming, the survival exceeded 80%. The percentages of langerhans cells and melanocytes in the primary passage were 5.8% and 8.1% respectively. In the 1st passage they were 2. 1% and 2. 8% respectively. They were not detected in the second passage. The values of cpm were respectively 482. 13±46.61 （primary passage）, 362.50±35.12（lst passage）, 228. 38±51.46（2nd passage）, 171.86±34.63（3rd passage）, 143.63±15.95（4th passage）, and 123.25±14.39（5th passage）, showing statistically significant differences when compared with control （53.67±8.61） （P〈0.05）. There were statistically significant differences between the primary passage, the 1st passage respectively and the other passages （P〈0. 05）. There were statistically significant differences between the 4th passage, the 5th passage respectively and the 2nd passage （P〈0. 05）. There was no statistically significant difference between the 2nd passage and the 3rd passages （P〉0.05）. There was not statistically significant difference among the 3rd, the 4th and the 5th passages （P〉0. 05）. Conclusion Allogenic keratinocytes were cultured in vitro and passaged, and their immunogenity gradually decreased.