条件性子宫细胞剔除的研究

Preliminary Study of Conditional Cell Depletion in Uterus

为了研究体内细胞的功能和各种细胞之间的相互作用,试验研究了可以特异性消除特定细胞的条件性细胞剔除新方法。通过PR^Cre/+工具鼠与pCAG-loxp-STOP-loxp-DTR-2A-EGFP转基因小鼠(DTR小鼠)交配获得子宫特异表达白喉毒素受体(DTR)的转基因小鼠,经过PCR和琼脂糖凝胶电泳鉴定基因后,筛选出PR^Cre/+/pCAG-loxp-STOP-loxp-DTR-2A-EGFP雌性小鼠(PR-DTR小鼠)。以DTR小鼠和野生型小鼠(WT小鼠)作为试验对照组,利用免疫组织化学和Western blotting检测子宫中绿色荧光蛋白(GFP)的表达。腹腔注射白喉毒素1 ng/d,连续2 d,观察PR-DTR小鼠、DTR小鼠和WT小鼠子宫的外部形态差异,并通过HE染色的方法观察子宫的内部形态。使用免疫组织化学方法检测注射白喉毒素后PR-DTR小鼠、DTR小鼠和WT小鼠子宫中叉头框蛋白A2(FOXA2)的表达。试验发现在小鼠子宫上皮、基质和肌层细胞中都有GFP的表达,注射白喉毒素后PR-DTR小鼠子宫红肿膨胀,DTR小鼠和WT小鼠子宫没有显著变化。HE染色试验结果发现,PR-DTR小鼠子宫中出现异常空腔,而DTR小鼠和WT小鼠子宫没有明显变化,并且对注射白喉毒素的PR-DTR小鼠子宫中FOXA2表达的检测结果表明,这些异常的空腔不属于腺体,说明白喉毒素使细胞死亡,证实在小鼠子宫中表达孕酮受体的细胞表达了DTR。试验结果表明,白喉毒素在PR-DTR小鼠子宫中可以条件性剔除子宫中表达孕酮受体的细胞,为研究子宫细胞的功能以及子宫细胞相互之间的作用提供了新方法。

In order to study the function of cells and the interaction between various cells in vivo,a new method of conditional cell depletion which could specifically eliminate specific cells was studied.Uterine-specific diphtheria toxin receptor transgenic mice were obtained by mating PR^Cre/+tool mice with pCAG-loxp-STOP-loxp-DTR-2A-EGFP transgenic mice(DTR mice).The genes were identified by PCR and agarose gel electrophoresis and PR^Cre/+/pCAG-loxp-STOP-loxp-DTR-2A-EGFP female mice(PR-DTR mice)were screened out.The DTR mice and wild type mice(WT mice)were used as experimental control groups.Immunohistochemistry and Western blotting were used to detect the expression of GFP protein in the uterus.1 ng/d diphtheria toxin was injected intraperitoneally to observe the differences in external morphology of the uterus of PR-DTR,DTR and wild type mice.The internal morphology of the uterus was observed by HE staining.Immunohistochemistry was used to detect the expression of FOXA2 protein in PR-DTR,DTR and WT female mice after injecting diphtheria toxin.It was found that epithelium,stromal and muscle cells of mouse expressed GFP protein.The uterus of PR-DTR mice was redness and swelling after injection of diphtheria toxin,and there were no significant changes in the uterus of DTR mice and WT mice.Abnormal cavities in the uterus of PR-DTR mice were observed by HE staining.There were no significant differences in the uterus of DTR and WT mice,the detection of FOXA2 expression in the uterus of PR-DTR mice injected with diphtheria toxin showed that these abnormal cavities were not glands,indicating that diphtheria toxin caused cell death.It was confirmed that cells expressing progesterone receptors in mouse uterus expressed DTR.Experiments showed that diphtheria toxin played a role in the uterus of PR-DTR mice,and could conditionally eliminate cells expressing progesterone receptors in the uterus.It provided a new method for studying the function of uterine cells and the interaction between uterine cells.