国产西罗莫司与原研品在体内外对细胞影响的比较实验

目的探讨国产西罗莫司与原研品对移植宿主外周血中免疫细胞的影响效果。方法体外实验:人膀胱癌T24细胞体外培养,分别加入国产西罗莫司和原研品,CKK-8法检测并比较细胞增殖活性受抑制的情况。体内实验:建立小鼠异位心脏移植模型,设立对照无手术组(对照组)、移植无治疗组(Tx组)、移植+国产西罗莫司组(Tx+YXK组)、移植+原研品组(Tx+RAPA组)。观察移植心脏搏动情况,受者脾脏的流式细胞学检测,以及脾脏及移植物中免疫细胞浸润的病理检查。流式细胞检测树突状细胞(DC),CD8+细胞和调节性T细胞(Treg),病理组织学检测及免疫组化染色比较两组免疫细胞浸润情况。两组间比较采用独立样本t检验,多组间比较采用单因素方差分析,两两比较采用LSD-t检验。结果体外实验结果显示,国产西罗莫司与原研品对T24细胞活力影响的差异无统计学意义(P>0.05)。体内实验结果显示,Tx组移植心脏于第7天停止搏动,Tx+YXK组和Tx+RAPA组在第10天心脏搏动仍有力、节律正常。(1)脾脏流式细胞检测显示,与对照组、Tx组比较,Tx+RAPA组、Tx+YXK组CD11c+I-A+CD86+DC细胞(15.88±4.73、22.90±3.86比4.51±1.57、5.40±2.54)、CD8+淋巴细胞数量(6.32±0.98、6.75±1.34比3.03±1.12、3.23±0.97)均降低,而Tx+RAPA组CD4+CD25+Foxp3+阳性细胞数量(15.06±3.42比7.87±1.95,10.88±2.08)升高(P均<0.05)。Tx+YXK组和Tx+RAPA组3种免疫细胞数量差异均无统计学意义(P>0.05)。(2)移植心脏病理免疫细胞组化染色灰度分析,Tx组、Tx+YXK组和Tx+RAPA组CD4,CD8,IDO和CD11b数量差异无统计学意义(P>0.05),与Tx组比较,Tx+RAPA组和Tx+YXK组CD11c(25143.52±3525.12比12936.30±766.94、14240.60±3124.67)、Foxp3阳性细胞浸润数量(500.78±238.33比46.05±68.16、49.22±25.82)降低(P均<0.05),Tx+YXK组和Tx+RAPA组比较差异无统计学意义(P>0.05)。(3)模型动物脾脏病理免疫细胞组化染色灰度分析,Tx组CD 4和CD8阳性细胞浸润数量较Tx+YXK组和Tx+RAPA组少,但差异无统计学意义(P>0.05),Tx+YXK组和Tx+RAPA组比较,各种细胞染色的IOD值差异均无统计学意义。结论使用国产西罗莫司与原研品两种药物后受者移植心脏和脾脏中的细胞浸润变化一致;在体外对细胞增殖、移植后抗排斥作用和体内免疫细胞的影响表现均一致。

Comparison the effects of domestic sirolimus and the original product on the cells in vitro and in vivo

ObjectiveTo investigate the effect of domestic sirolimus (yixinke) and the original product Rapamycin on the cytology of transplanted host in vitro and in vivo. MethodsT24 cells were cultured in vitro, and Yixinke and Rapamycin were treated respectively. The cell proliferation was rate assessed by CKK-8 kit. An in vivo model of heterotopic heart transplantation in mice was established, and the mice were divided into a control group (without surgery) , a transplantation group (Tx group without therapy) , Yixinke treatment group (Tx+YXK group) and Rapamycin treatment group (Tx+RAPA group) . Beating of the transplanted heart, the composition of the recipient's spleen cells, and infiltration of immune cells into the spleen and the grafts were monitored. The main immune cells detected by flow cytometry were dendritic cells, CD8⁺ T cells and regulatory T cells. Histopathological examination, immunohistochemical staining and gray level difference analysis were used to compare the infiltration of immune cells between the two treatment groups. The ANOVA and two-independent t-tests were used for statistical analysis. ResultsIn vitro experiments showed that the effect of domestic sirolimus on the proliferation of T24 cells was the same as that of the original product (P > 0.05) . In vivo experiments showed that the beating of the transplanted heart in the Tx group stopped on the 7th day, and in the Tx+YXK group and Tx+ RAPA group, the beating of the transplanted heart was still normal and strong on the 10th day. (1) Flow cytometric analysis of spleen revealed that compared with the control group and Tx group, the number of CD11c⁺I- A⁺CD86⁺DC cells and CD8⁺ lymphocytes (15.88±4.73, 22.90±3.86 vs 4.51±1.57, 5.40±2.54, 6.32±0.98, 6.75±1.34 vs 3.03±1.12, 3.23±0.97) in Tx+RAPA group and Tx+YXK group were all decreased (P < 0.05) , and the number of CD4⁺CD25⁺Foxp3⁺ positive cells in the Tx+ RAPA group increased (P < 0.05) . There was no significant difference in the number of CD11c⁺I-A⁺CD86⁺DC, CD8⁺ lymphocytes, CD4⁺CD25⁺Foxp3⁺ regulatory T cells between Tx+YXK group and Tx+Rapa group (P > 0.05) . (2) According to immunohistochemical staining and gray level difference analysis for the transplanted heart, there was no significant difference in the number of CD4, CD8, Ido and CD11b between Tx group, Tx+YXK group and Tx+Rapa group (P > 0.05) . Compared with Tx group, the number of CD11c and Foxp3 positive cells infiltrated in Tx+RAPA group and Tx+YXK group (25143.52±3525.12 vs 12936.30±766.94, 14240.60±3124.67, 500.78±238.33 vs 46.05±68.16, 49.22±25.82) was lower. There was no significant difference between Tx+YXK group and Tx+Rapa group (P > 0.05) . (3) Immunohistochemical staining and gray level difference analysis for the spleen of the model mice showed that the number of CD4 and CD8 positive cells infiltrated in the Tx group was less than that in the Tx+YXK group and Tx+RAPA group, but the difference was not statistically significant (P > 0.05) . Between the Tx+YXK group and Tx+Rapa group, the difference of IOD value of various cell staining was not statistically significant. ConclusionOur results showed that cell infiltration into the transplanted heart and spleen in the Yixinke and conventional drug treatment groups was similar. The effects of Yixinke and the conventional drug on cell proliferation in vitro, anti-rejection following transplantation and immune cells in vivo were all similar.