目的:探讨过表达MARCH2对肝细胞癌HUH7细胞自噬水平和生长的影响及其机制。方法:MTS和EDU检测MARCH2过表达的肝细胞癌HUH7细胞的生长情况。流式细胞术检测MARCH2过表达的肝细胞癌HUH7细胞凋亡、细胞周期的情况。Western blot和激光共聚...目的:探讨过表达MARCH2对肝细胞癌HUH7细胞自噬水平和生长的影响及其机制。方法:MTS和EDU检测MARCH2过表达的肝细胞癌HUH7细胞的生长情况。流式细胞术检测MARCH2过表达的肝细胞癌HUH7细胞凋亡、细胞周期的情况。Western blot和激光共聚焦法检测MARCH2过表达的肝细胞癌HUH7细胞自噬水平。结果:MTS和EDU检测表明,MARCH2的过表达促进了肝细胞癌HUH7的生长。流式细胞术检测表明,MARCH2过表达对肝细胞癌HUH7细胞凋亡和细胞周期没有明显影响。进一步的实验表明,MARCH2过表达损害了肝细胞癌HUH7的自噬。用常见的自噬诱导剂雷帕霉素挽救自噬可以使肝细胞癌HUH7的增殖率恢复到控制水平。结论:我们的数据表明,MARCH2通过抑制自噬在肿瘤发展过程中起着至关重要的作用。因此,MARCH2是一种有前景的肿瘤治疗新靶点。Aims: To investigate the effects and mechanisms of overexpression of MARCH2 on autophagy levels and growth of hepatocellular carcinoma HUH7 cells. Methods: MTS and EDU were used to detect the growth of MARCH2 overexpressing hepatocellular carcinoma HUH7 cells. Flow cytometry was used to detect apoptosis and cell cycle in MARCH2 overexpressing hepatocellular carcinoma HUH7 cells. Western blot and laser confocal microscopy were used to detect the autophagy levels in MARCH2 overexpressing HUH7 hepatocellular carcinoma cells. Results: In this study, MTS and EDU assays showed that the overexpression of MARCH2 promoted the growth of hepatocellular carcinoma HUH7 cells. Flow cytometry detection illustrated that MARCH2 overexpression had no obvious effect on the hepatocellular carcinoma HUH7 cell apoptosis and cell cycle. Further experiments displayed that MARCH2 overexpression impaired the autophagy of hepatocellular carcinoma HUH7 cells. Rescued autophagy with the common autophagy inducer rapamycin can bring the proliferation rate of hepatocellular carcinoma HUH7 cells back to the control level. Conclusions: Our data indicates that MARCH2 plays an essential role in the progression of tumor development through inhibiting autophagy. Therefore, MARCH2 is an attractive novel target for tumor therapy.展开更多
目的:探讨MARCH2和CFTR在结直肠癌中的蛋白表达及与临床病理参数的相关性。方法:采用免疫组织化学SP法检测125例结直肠癌和30例正常结直肠黏膜组织中MARCH2和CFTR的表达,并复习相关文献。结果:在125例结直肠癌组织中,MARCH2和CFTR的阳...目的:探讨MARCH2和CFTR在结直肠癌中的蛋白表达及与临床病理参数的相关性。方法:采用免疫组织化学SP法检测125例结直肠癌和30例正常结直肠黏膜组织中MARCH2和CFTR的表达,并复习相关文献。结果:在125例结直肠癌组织中,MARCH2和CFTR的阳性表达率分别为84.8%和8.8%,而在非肿瘤性肠组织中,这两种表达率的阳性率分别为6.7%和90.0%。MARCH2的阳性表达与肿瘤大小、深部浸润、淋巴结转移和TNM分期密切相关。此外,CFTR表达的缺失与分化不良、淋巴结转移和TNM分期显著相关。此外,MARCH2和CFTR表达之间存在显著负相关。结论:MARCH2在结直肠癌中高度表达,与肿瘤大小、深部浸润、淋巴结转移和TNM分期等预后不良参数有关。相比之下,CFTR在结直肠癌中几乎不表达,CFTR表达缺失与分化不良、淋巴结转移和TNM分期显著相关。MARCH2可能通过与CFTR的相互作用在结直肠癌的发展中发挥重要作用。它们可能成为结直肠癌诊断、治疗和预后评估的分子标志物。Aims: This study aimed to investigate whether the expressions of MARCH2 and CFTR in colorectal carcinomas were associated with clinicopathological parameters. Methods: The expressions of MARCH2 and CFTR were examined in 125 cases of colorectal carcinomas and 30 normal colorectal tissues using immunohistochemical staining. Results: The positive rates of MARCH2 and CFTR expressions in 125 cases of colorectal carcinoma were 84.8% and 8.8% respectively, whereas the positive expression rates of MARCH2 and CFTR in non-tumor colorectal tissues were 6.7% and 90.0% respectively. The positive expressions of MARCH2 were closely related to tumor size, deep invasion, lymph node metastasis and advanced TNM stage. In addition, loss of expression of CFTR was significantly associated with poor differentiation, lymph node metastasis and advanced TNM stage. Furthermore, there were significant negative correlations between MARCH2 and CFTR expression. Conclusions: MARCH2 is highly expressed in colorectal carcinomas, and is associated with poor prognostic parameters such as tumor size, deep invasion, lymph node metastasis and advanced TNM stage. In contrast, CFTR barely expressed in colorectal carcinomas, and loss of expression of CFTR was significantly associated with poor differentiation, lymph node metastasis and advanced TNM stage. MARCH2 may play an important role in the development of colorectal carcinoma through interaction with CFTR. They may become molecular markers for diagnosis, treatment and prognostic evaluation of colorectal carcinomas.展开更多
目的:探讨MARCH2多克隆抗体的制备、鉴定与纯化的方法以及其在组织和细胞系中的表达情况、在亚细胞结构中的定位情况。方法:利用DNAstar软件对MARCH2蛋白的抗原性等进行分析,化学合成MARCH2短肽,制备成完全抗原免疫家兔,获取血清,纯化,...目的:探讨MARCH2多克隆抗体的制备、鉴定与纯化的方法以及其在组织和细胞系中的表达情况、在亚细胞结构中的定位情况。方法:利用DNAstar软件对MARCH2蛋白的抗原性等进行分析,化学合成MARCH2短肽,制备成完全抗原免疫家兔,获取血清,纯化,用Western blot、Elisa、免疫荧光进行鉴定。用RT-PCR检测MARCH2在细胞系中的表达情况,用Western blot检测MARCH2在组织中的表达情况。用免疫荧光法及激光共聚焦显微镜分析检测MARCH2在亚细胞结构中的定位。结果:成功制备完全抗原免疫家兔,纯化出多克隆抗体,用Western blot、Elisa、免疫荧光法证实多克隆抗体制备成功。利用半定量RT-PCR方法,在32种细胞系中检测了MARCH2 mRNA的表达水平,结果显示,MARCH2呈广泛表达,在HeLa、U2OS、HCT116、COS7细胞中表达最高,在SKBR3、HGC-27、MGC-803细胞中低表达。利用组织芯片及免疫组织化学方法进一步分析了其在正常组织及肿瘤组织中的表达情况,染色结果显示,MARCH2呈广泛表达,在前列腺、肝组织中呈高表达,在横纹肌、甲状腺组织中呈低表达,主要在胞浆中呈现弥漫均匀细颗粒状分布。此外,MARCH2表达因肿瘤类型的不同有差异。与对应的正常组织相比,MARCH2在某些肿瘤(如前列腺癌、肝癌)组织中表达降低,而在某些肿瘤(如食管癌、结肠癌)组织中表达增高。用免疫荧光法及激光共聚焦显微镜分析检测MARCH2在亚细胞结构中的定位,发现MARCH2与内质网、高尔基体共定位,与溶酶体、内体部分共定位,与线粒体没有共定位。结论:成功获得了MARCH2多克隆抗体,为进一步研究MARCH2蛋白的功能奠定了基础。MARCH2在不同类型肿瘤中的差异表达及其在亚细胞结构中的定位高度提示MARCH2在肿瘤发生发展中具有重要的潜在应用价值。Aims: To explore the preparation, identification, and purification methods of MARCH2 polyclonal antibodies, as well as their expression and subcellular structural localization in tissues and cell lines. Methods: DNAstar software was used to analyze the antigenicity of MARCH2 protein, and MARCH2 short peptides were chemically synthesized to prepare complete antigen-immunized rabbits. Serum was obtained, purified, and identified by Western blot, Elisa, and immunofluorescence. RT-PCR was used to detect the expression of MARCH2 in cell lines, and Western blot was used to detect the expression of MARCH2 in tissues. Immunofluorescence and confocal laser microscopy were used to analyze and detect the localization of MARCH2 in subcellular structures. Results: Fully antigen-immunized rabbits were successfully prepared, and polyclonal antibodies were purified. Western blot, Elisa, and immunofluorescence were used to confirm the successful preparation of polyclonal antibodies. Using semi-quantitative RT-PCR method, the expression level of MARCH2 mRNA was detected in 32 cell lines. The results showed that MARCH2 was widely expressed, with the highest expression in HeLa, U2OS, HCT116, and COS7 cells, and low expression in SKBR3, HGC-27, and MGC-803 cells. The expression of MARCH2 in normal and tumor tissues was further analyzed using tissue chips and immunohistochemical methods. The staining results showed that MARCH2 was widely expressed, with high expression in prostate and liver tissues, low expression in striated muscle and thyroid tissues, and mainly distributed in a diffuse and uniform granular form in the cytoplasm. In addition, MARCH2 expression varies depending on the type of tumor. Compared with corresponding normal tissues, MARCH2 expression is reduced in certain tumor tissues (such as prostate cancer and liver cancer), while it is increased in certain tumor tissues (such as esophageal cancer and colon cancer). Using immunofluorescence and laser confocal microscopy to analyze and detect the localization of MARCH2 in subcellular structures, it was found that MARCH2 was co-localized with the endoplasmic reticulum and Golgi apparatus, with lysosomes and endosomes partially, but not with mitochondria. Conclusions: MARCH2 polyclonal antibodies have been successfully obtained, laying the foundation for further research on the function of MARCH2 protein. The differential expression of MARCH2 in different types of tumors and its localization in subcellular structures highly suggest that MARCH2 has important potential application value in tumor occurrence and development.展开更多
文摘目的:探讨过表达MARCH2对肝细胞癌HUH7细胞自噬水平和生长的影响及其机制。方法:MTS和EDU检测MARCH2过表达的肝细胞癌HUH7细胞的生长情况。流式细胞术检测MARCH2过表达的肝细胞癌HUH7细胞凋亡、细胞周期的情况。Western blot和激光共聚焦法检测MARCH2过表达的肝细胞癌HUH7细胞自噬水平。结果:MTS和EDU检测表明,MARCH2的过表达促进了肝细胞癌HUH7的生长。流式细胞术检测表明,MARCH2过表达对肝细胞癌HUH7细胞凋亡和细胞周期没有明显影响。进一步的实验表明,MARCH2过表达损害了肝细胞癌HUH7的自噬。用常见的自噬诱导剂雷帕霉素挽救自噬可以使肝细胞癌HUH7的增殖率恢复到控制水平。结论:我们的数据表明,MARCH2通过抑制自噬在肿瘤发展过程中起着至关重要的作用。因此,MARCH2是一种有前景的肿瘤治疗新靶点。Aims: To investigate the effects and mechanisms of overexpression of MARCH2 on autophagy levels and growth of hepatocellular carcinoma HUH7 cells. Methods: MTS and EDU were used to detect the growth of MARCH2 overexpressing hepatocellular carcinoma HUH7 cells. Flow cytometry was used to detect apoptosis and cell cycle in MARCH2 overexpressing hepatocellular carcinoma HUH7 cells. Western blot and laser confocal microscopy were used to detect the autophagy levels in MARCH2 overexpressing HUH7 hepatocellular carcinoma cells. Results: In this study, MTS and EDU assays showed that the overexpression of MARCH2 promoted the growth of hepatocellular carcinoma HUH7 cells. Flow cytometry detection illustrated that MARCH2 overexpression had no obvious effect on the hepatocellular carcinoma HUH7 cell apoptosis and cell cycle. Further experiments displayed that MARCH2 overexpression impaired the autophagy of hepatocellular carcinoma HUH7 cells. Rescued autophagy with the common autophagy inducer rapamycin can bring the proliferation rate of hepatocellular carcinoma HUH7 cells back to the control level. Conclusions: Our data indicates that MARCH2 plays an essential role in the progression of tumor development through inhibiting autophagy. Therefore, MARCH2 is an attractive novel target for tumor therapy.
文摘目的:探讨MARCH2和CFTR在结直肠癌中的蛋白表达及与临床病理参数的相关性。方法:采用免疫组织化学SP法检测125例结直肠癌和30例正常结直肠黏膜组织中MARCH2和CFTR的表达,并复习相关文献。结果:在125例结直肠癌组织中,MARCH2和CFTR的阳性表达率分别为84.8%和8.8%,而在非肿瘤性肠组织中,这两种表达率的阳性率分别为6.7%和90.0%。MARCH2的阳性表达与肿瘤大小、深部浸润、淋巴结转移和TNM分期密切相关。此外,CFTR表达的缺失与分化不良、淋巴结转移和TNM分期显著相关。此外,MARCH2和CFTR表达之间存在显著负相关。结论:MARCH2在结直肠癌中高度表达,与肿瘤大小、深部浸润、淋巴结转移和TNM分期等预后不良参数有关。相比之下,CFTR在结直肠癌中几乎不表达,CFTR表达缺失与分化不良、淋巴结转移和TNM分期显著相关。MARCH2可能通过与CFTR的相互作用在结直肠癌的发展中发挥重要作用。它们可能成为结直肠癌诊断、治疗和预后评估的分子标志物。Aims: This study aimed to investigate whether the expressions of MARCH2 and CFTR in colorectal carcinomas were associated with clinicopathological parameters. Methods: The expressions of MARCH2 and CFTR were examined in 125 cases of colorectal carcinomas and 30 normal colorectal tissues using immunohistochemical staining. Results: The positive rates of MARCH2 and CFTR expressions in 125 cases of colorectal carcinoma were 84.8% and 8.8% respectively, whereas the positive expression rates of MARCH2 and CFTR in non-tumor colorectal tissues were 6.7% and 90.0% respectively. The positive expressions of MARCH2 were closely related to tumor size, deep invasion, lymph node metastasis and advanced TNM stage. In addition, loss of expression of CFTR was significantly associated with poor differentiation, lymph node metastasis and advanced TNM stage. Furthermore, there were significant negative correlations between MARCH2 and CFTR expression. Conclusions: MARCH2 is highly expressed in colorectal carcinomas, and is associated with poor prognostic parameters such as tumor size, deep invasion, lymph node metastasis and advanced TNM stage. In contrast, CFTR barely expressed in colorectal carcinomas, and loss of expression of CFTR was significantly associated with poor differentiation, lymph node metastasis and advanced TNM stage. MARCH2 may play an important role in the development of colorectal carcinoma through interaction with CFTR. They may become molecular markers for diagnosis, treatment and prognostic evaluation of colorectal carcinomas.
文摘目的:探讨MARCH2多克隆抗体的制备、鉴定与纯化的方法以及其在组织和细胞系中的表达情况、在亚细胞结构中的定位情况。方法:利用DNAstar软件对MARCH2蛋白的抗原性等进行分析,化学合成MARCH2短肽,制备成完全抗原免疫家兔,获取血清,纯化,用Western blot、Elisa、免疫荧光进行鉴定。用RT-PCR检测MARCH2在细胞系中的表达情况,用Western blot检测MARCH2在组织中的表达情况。用免疫荧光法及激光共聚焦显微镜分析检测MARCH2在亚细胞结构中的定位。结果:成功制备完全抗原免疫家兔,纯化出多克隆抗体,用Western blot、Elisa、免疫荧光法证实多克隆抗体制备成功。利用半定量RT-PCR方法,在32种细胞系中检测了MARCH2 mRNA的表达水平,结果显示,MARCH2呈广泛表达,在HeLa、U2OS、HCT116、COS7细胞中表达最高,在SKBR3、HGC-27、MGC-803细胞中低表达。利用组织芯片及免疫组织化学方法进一步分析了其在正常组织及肿瘤组织中的表达情况,染色结果显示,MARCH2呈广泛表达,在前列腺、肝组织中呈高表达,在横纹肌、甲状腺组织中呈低表达,主要在胞浆中呈现弥漫均匀细颗粒状分布。此外,MARCH2表达因肿瘤类型的不同有差异。与对应的正常组织相比,MARCH2在某些肿瘤(如前列腺癌、肝癌)组织中表达降低,而在某些肿瘤(如食管癌、结肠癌)组织中表达增高。用免疫荧光法及激光共聚焦显微镜分析检测MARCH2在亚细胞结构中的定位,发现MARCH2与内质网、高尔基体共定位,与溶酶体、内体部分共定位,与线粒体没有共定位。结论:成功获得了MARCH2多克隆抗体,为进一步研究MARCH2蛋白的功能奠定了基础。MARCH2在不同类型肿瘤中的差异表达及其在亚细胞结构中的定位高度提示MARCH2在肿瘤发生发展中具有重要的潜在应用价值。Aims: To explore the preparation, identification, and purification methods of MARCH2 polyclonal antibodies, as well as their expression and subcellular structural localization in tissues and cell lines. Methods: DNAstar software was used to analyze the antigenicity of MARCH2 protein, and MARCH2 short peptides were chemically synthesized to prepare complete antigen-immunized rabbits. Serum was obtained, purified, and identified by Western blot, Elisa, and immunofluorescence. RT-PCR was used to detect the expression of MARCH2 in cell lines, and Western blot was used to detect the expression of MARCH2 in tissues. Immunofluorescence and confocal laser microscopy were used to analyze and detect the localization of MARCH2 in subcellular structures. Results: Fully antigen-immunized rabbits were successfully prepared, and polyclonal antibodies were purified. Western blot, Elisa, and immunofluorescence were used to confirm the successful preparation of polyclonal antibodies. Using semi-quantitative RT-PCR method, the expression level of MARCH2 mRNA was detected in 32 cell lines. The results showed that MARCH2 was widely expressed, with the highest expression in HeLa, U2OS, HCT116, and COS7 cells, and low expression in SKBR3, HGC-27, and MGC-803 cells. The expression of MARCH2 in normal and tumor tissues was further analyzed using tissue chips and immunohistochemical methods. The staining results showed that MARCH2 was widely expressed, with high expression in prostate and liver tissues, low expression in striated muscle and thyroid tissues, and mainly distributed in a diffuse and uniform granular form in the cytoplasm. In addition, MARCH2 expression varies depending on the type of tumor. Compared with corresponding normal tissues, MARCH2 expression is reduced in certain tumor tissues (such as prostate cancer and liver cancer), while it is increased in certain tumor tissues (such as esophageal cancer and colon cancer). Using immunofluorescence and laser confocal microscopy to analyze and detect the localization of MARCH2 in subcellular structures, it was found that MARCH2 was co-localized with the endoplasmic reticulum and Golgi apparatus, with lysosomes and endosomes partially, but not with mitochondria. Conclusions: MARCH2 polyclonal antibodies have been successfully obtained, laying the foundation for further research on the function of MARCH2 protein. The differential expression of MARCH2 in different types of tumors and its localization in subcellular structures highly suggest that MARCH2 has important potential application value in tumor occurrence and development.
