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目的:建立稳定的ICC分离培养方法,明确L-Ca~(2+)通道CaV1.3在ICC的表达。方法:取出生1—2天正常的BALB/c小鼠小肠,Ⅱ型胶原酶消化,过筛网、梯度离心后接种至铺有鼠尾胶原的培养瓶中培养。RT-PCR技术检测ICC特异性标志c-kit mRNA的表达情况;免疫荧光检冽c-kit蛋白的表达和定位;流式细胞术检测ICC纯度。RT-PCR技术检测CaV1.3 mRNA的表;免疫荧光双染检测c-kit蛋白和CaV1.3蛋白在ICC的定位。结果;培养的ICC贴壁细胞多角形,核大,细胞突起形成网状连接。RT-PCR和免疫荧光技术检测到ICC特异性标志c-kit mRNA和c-kit蛋白的表达。流式细胞术所得ICC纯度为81.97%。结论:运用Ⅱ型胶原酶消化-密度梯度离心法,我们建立了稳定的ICC培养方法,获得了相对较纯的单一细胞,为ICC进一步的基础研究奠定了坚实的基础。L-Ca~(2+)通道CaV1.3在ICC细胞膜大量表达,与ICC起搏功能存在相关性。
OBJECTIVE: To establish a stable ICC culture method to determine the expression of L-Ca 2+ channel CaC1.3 in ICC. Methods: The small intestine of normal BALB / c mice born 1-2 days old was digested and digested with type Ⅱ collagenase. After being sieved through a sieve, the cells were centrifuged and then inoculated into culture flasks coated with rat tail collagen. The expression of ICC-specific marker c-kit mRNA was detected by RT-PCR. The expression of c-kit protein was detected by immunofluorescence and the purity of ICC was detected by flow cytometry. The expression of CaV1.3 mRNA was detected by RT-PCR. The localization of c-kit protein and CaV1.3 protein in ICC was detected by double immunofluorescence staining. Results; ICC adherent cells cultured polygons, large nucleus, cell processes to form a mesh connection. The expression of ICC-specific marker c-kit mRNA and c-kit protein was detected by RT-PCR and immunofluorescence. The ICC purity obtained by flow cytometry was 81.97%. CONCLUSION: By using type Ⅱ collagenase digestion-density gradient centrifugation, we established a stable ICC culture method and obtained a relatively pure single cell, which laid a solid foundation for further basic research of ICC. L-Ca 2+ channel CaV1.3 is highly expressed in the ICC cell membrane, which is correlated with ICC pacing function.