【作者】 董丹；

【导师】 戈应滨；

【作者基本信息】 南京医科大学 ， 生理学， 2010， 硕士

【摘要】 背景Cajal间质细胞（interstitial cell of Cajal,ICC）被公认为胃肠道慢波的起搏细胞。ICC起搏机制的研究是目前ICC研究的一大热点,其中钙离子(Ca²⁺)贯穿ICC起搏活动的全过程, ICC钙瞬变（calcium transient）快速上升期中钙星（calcium sparklet）的形成通道是争论的焦点。用膜片钳方法记录到单个ICC最大的Ca²⁺流是通过L-Ca²⁺通道形成,而二氢吡啶类Ca²⁺通道拮抗剂并不改变ICC慢波曲线。其他细胞的研究表明, CaV1.3是一类对二氢吡啶类相对不敏感的L-Ca²⁺通道。我们推测,L-Ca²⁺通道CaV1.3可能在ICC中表达,并且参与了ICC起搏活动。目的1.建立稳定的ICC分离,培养方法。2.明确L-Ca²⁺通道CaV1.3在ICC的表达情况,探讨L-Ca²⁺通道CaV1.3与ICC起搏功能的关系。方法分为两大部分:第一部分:ICC培养和鉴定ICC培养:取出生1-2d正常的BALB/c小鼠小肠,置于1.0mg/mlⅡ型胶原酶消化,过200目筛网去除大块组织,密度梯度离心法筛选细胞,接种至铺有鼠尾胶原的培养瓶,37℃,5%CO2条件下,M199培养基中培养。ICC鉴定:RT-PCR技术检测ICC特异性标志c-kit mRNA的表达情况;免疫荧光检测c-kit蛋白的表达和定位;流式细胞术检测ICC纯度;c-kit蛋白和EdU免疫荧光双染检测ICC增殖情况。第二部分:L-Ca²⁺通道CaV1.3在ICC的表达和肌张力变化的关系L-Ca²⁺通道CaV1.3表达:RT-PCR技术检测CaV1.3 mRNA的表达情况;免疫荧光双染检测c-kit蛋白和CaV1.3蛋白在ICC的定位。取出生0d,7d,14d和成年BALB/c小鼠小肠,进行c-kit蛋白和CaV1.3蛋白免疫组化染色,检测不同发育期两种蛋白的共表达变化情况。同时检测对各个时间段的小肠的肌张力,探讨肌张力的变化和CaV1.3蛋白的关系。结果第一部分:ICC培养和鉴定培养的ICC贴壁细胞多角形,核大,细胞突起形成网状连接。ICC存活10d以上,可见细胞数量的增多。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%。EdU染色结果显示EdU与c-kit阳性细胞共表达。第二部分:L-Ca²⁺通道CaV1.3在ICC的表达和肌张力变化的关系RT-PCR检测到CaV1.3 mRNA在培养细胞中表达;免疫荧光双染显示c-kit蛋白和CaV1.3蛋白在ICC细胞共表达。出生0d,7d,14dBALB/c小鼠小肠的c-kit蛋白和CaV1.3蛋白免疫组化染色结果显示,两种蛋白共表达数量逐渐增加,出生14d时已接近成年小鼠小肠免疫组化结果。各个时间段小肠肌张力测定结果表明,出生0d,7d,14d小肠收缩曲线逐渐趋于规整,出生14d时已接近成年小鼠小肠收缩曲线结果。CaV1.3表达量和小肠收缩曲线呈正相关。结论1.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,获得了相对纯化的单一细胞,为ICC进一步的基础研究奠定了坚实的基础。2.体外培养的ICC具有增殖能力。3. L-Ca²⁺通道CaV1.3在ICC细胞膜大量表达,与ICC起搏功能存在正相关性。更多还原

【Abstract】 BackgroundThe interstitial cell of Cajal （ICC） is found in the gastrointestinal tract and serveed as a pacemaker, in which create the basal electrical rhythm. Abnormality of ICC pacemaker functions lead to gastrointestinal motility disorder, such as chronic intestinal pseudo-obstruction （CIP）, diabetic gastroparesis and so on. Ca2+ exists throughout the whole pacemaker time. The channels of calcium sparklet formation are the point at issue in fast-rise phase of calcium transient. In patch clamp recordings of single human intestinal ICC, the largest Ca2+ current is a Ca2+ current carried by L-type Ca2+ channel. Previous studies have suggested pacemaker activity in ICC is associated with robust Ca2+ entry events that are resistant to block by dihydropyridines. And L-type CaV1.3 Ca2+ channel is relatively insensitive to dihydropyridines. We suppose that L-type CaV1.3 Ca2+ channel expressed in ICC, and may be functionally important for the pacemaker activity in ICC, maybe inducing the formation of calcium sparklet.Objective1. Establish a stable method of ICC isolation and cell culture2. Identify the expression of L-type CaV1.3 Ca2+ channel in ICC, and the relation between L-type CaV1.3 Ca2+ channel and pacemaker activity.MethodsPart 1: Cell culture and identificationCell culture: small intestine muscle pieces from BALB/c mice （1-2 d old） were incubated in solution containing 1.0mg/ml collagenase （type II）. After passing through the sieve （size: #200）, all cell suspension was layered on the surface of a 200 g/L Ficoll density cushion. The cell band located at the interface was plated into dishes with collagen-coated bottoms. The cells resuspended with M199 medium were maintained in 50% CO2 at 37℃.Cell identification: reverse-transcriptase PCR （RT-PCR） was used to detect expression of c-kit mRNA; immunofluorescent staining was used to identify cell location of c-kit; flow cytometry was used to determine purity of cultured ICC; double-immunofluorescent staining was performed to detect ability of proliferation in vitro.Part 2: Expression of L-type CaV1.3 Ca²⁺ channel in ICCExpression of CaV1.3: RT-PCR was used to detect expression of CaV1.3 mRNA; double-immunofluorescent staining was used to detect cell location of c-kit and CaV1.3 in cultured ICC. Small intestine muscle pieces from BALB/c mice （postnatal day 0, 7, 14 and adult） were used in this study. Double-immunofluorescent staining was used to detect cell location of c-kit and CaV1.3. Spontaneous contractile activity was recorded to determine the functional changes of small intestine.ResultPart 1: Cell culture and identificationThe cultured adhesive cells were multangular with large nuclei in the centre and a network was formed by cytoplasmic processes. Cells were alive for more than 10 days, with an increase in the number. C-kit mRNA and protein were detected in cultured ICC by methods of RT-PCR and immunofluorescent staining. The purity was 81.97% using flow cytometry. Result of EdU staining directly showed that there was coexpression of EdU and c-kit protein. Part 2: Expression of L-type CaV1.3 Ca²⁺ channel in ICCCaV1.3 mRNA was detected in cultured cells. C-kit and CaV1.3 proteins coexpressed in ICC cell membrane. Double-immunofluorescent staining of the four growth periods showed that coexpression of the two proteins increased gradually in intensity and the intensity of coexpression from P14 was close to that from adult. The contraction curves became more regular with the processing of growth. Frequency and range of curve from P14 were nearly the same as that from adult.Conclusions1. Using Collagenase - Ficoll method, we established a stable ICC cell culture method, which lays a solid foundation for the basic and clinical research in ICC.2. ICC cultured in vitro had the ability to proliferate.3. L-type CaV1.3 Ca²⁺ channel was abundantly expressed in cell membrane of ICC, and there was a correlation between L-type CaV1.3 Ca²⁺ channel and ICC pacemaker activity.更多还原