核心蛋白聚糖抑制淋巴细胞增殖及皮肤移植免疫排斥反应的研究

The inhibitory effect of decorin on lymphocyte proliferation and immunological rejection in skin transplantation

目的 观察核心蛋白聚糖（DCN）对小鼠皮肤移植模型术后免疫排斥反应的影响.方法 以Ficoll密度梯度离心法提取C57BL/6J脾脏淋巴细胞,96孔板接种后,各孔加入25μg刀豆蛋白A.将细胞分为5组,分别加入0、25、50、75、100μg/L的DCN,同时设立仅含细胞悬液阴性对照组和培养液空白对照组.各组细胞培养24、48、72、96、120 h后,以噻唑蓝（MTT）比色法检测各组淋巴细胞增殖率,并以流式细胞仪检测各组细胞凋亡率.以24只健康成年BALB/C小鼠为供体,30只健康成年C57BL/6J小鼠为受体,将受体随机分为5组,建立背部皮肤移植模型.A组为阴性对照组,术后腹腔注射生理盐水1 ml;B组、C、D组受体小鼠于术后分别腹腔注射50、100、150μg的DCN.同时设立E组,为C57BL/6J对C57BL/6J的同种同基因移植对照组.观察各组受体移植皮片的存活时间和急性排斥反应发生时间.术后第7天,每组各取1只受体小鼠的移植皮片行病理学检查.结果 各组不同浓度DCN对小鼠脾脏淋巴细胞体外增殖均有抑制作用,并可诱导淋巴细胞凋亡,随DCN浓度升高对淋巴细胞增殖抑制作用更强,100μg/L组抑制作用最明显,细胞凋亡率为77.6%,与其他各组比较差异有统计学意义（P＜0.05）;DCN具有抑制皮肤移植免疫排斥反应及延长移植皮片存活时间,150μg组皮片存活时间最长,平均为（16.6±2.4）d,与其他各组比较差异有统计学意义（P＜0.05）.且随剂量增大抑制作用明显,移植皮肤急性排斥反应轻.结论 DCN具有免疫抑制作用,可能通过诱导活化的淋巴细胞凋亡抑制小鼠皮肤移植术后急性排斥反应发生,并延长移植皮片的存活时间.

Objective To study the inhibitory effect of decorin （DCN） on immunological rejection in skin transplantation.Methods Spleen lymphocytes were obtained from C57BL/6J mice,inoculated in a 96-well plate,and every well was added with 25 μg CoA.These cells were divided into 5 groups and cultured with DCN in different concentrations （0,25,50,75,100 μg/L） for different time （24,48,72,96 and 120 h）.Negative control group （lymphocytes only） and blank control group （culture medium only）were set up.Cell proliferation rate was measured by methylthiazol tetrazolium （MTT） assay,and the percentage of cell apoptosis was analyzed by flow cytometry.Twenty-four healthy BALB/C mice served as donors.Thirty C57BL/6J mice served as recipients and divided into 5 groups.Full-thickness-skin was transplanted through back to back from donor BALB/C to recipient C57BL/6J.Postoperatively,the recipients in group A （negative control） were intraperitoneally injected with 0.9% NaCl,and those in groups B,C and D with DCN （50,100 and 150 μg/L respectively）.In group F skin transplantation was performed between two C57BL/6J mice.After skin transplantation,the survival time of skin allografts and the time of acute rejection were observed.Skin specimens were collected from one recipient of every group 7 days after operation and examined pathologically.Results DCN obviously depressed mice spleen lymphocyte proliferation in vitro and inducef lymphocyte apoptosis in vitro.In 100 μg/L DCN group （ group C ）,there were most significant inhibitory effects,and the apoptosis rate was 77.6%.There were significant difference between group C and other groups （ P〈0.05 ）.DCN could inhibit immunological rejection of skin transplantation and prolong the survival time of skin allografts.Among all the groups,survival time in group D was longest with the average survival time being （ 16.6 ±2.4） days （ P 〈0.05 ）.The inhibitory effect of DCN was increased and the acute rejection was alleviated with the increase of DCN dosage.Conclusion DCN has the inhibitory effect.It can induce lymphocyte apoptosis and inhibit acute immunological rejection in skin transplantation.It can prolong the survival time of skin grafts.