| Cervical cancer is one of the most common neoplastic diseases affecting women.Infection with some genotypes of human papillomavirus(HPV)is the most important risk factor associated to cervical cancer.The causal relationship between chronic inflammation and cancer is widely acceptedz.Macrophages are the most existed inflammatory cells in tumor tissue.All macrophages existed in tumor tissue are collectively called as TAM.TAM was divided into two types according to the status and functions of macrophage activation including classical activation type(M1)and alternative activation type(M2).Ml macrophages can promote tumor immunity and primarily used for inhibiting tumor.M2 macrophage function was to promote tumor growth and metastasis.Tim-3 is the member of the Tim family.There are something relationship between M1-TAM,M2-TAM and Tim-3 with the tumor prognosis,but what the role the TAM polarization and Tim-3 palyed in the cervical cancer is not clear.For the purpose to explan the function of TAM and Tim-3 in cervical tumor microenvironment,all of the following will be study:(1)Detecting the expression of M1-TAM,M2-TAM and Tim-3 in cervical carcinoma and paracancerous tissue samples.And their collaborative relationship with stages of cervical cancer transfer to or not to Lymph nodes,surving times and the prognosis of patients.(2)Analysis of the effection of cervical tumor microenvironment to differentiation of TAM and Tim-3.(3)Analysis of Tim-3 and TAM polarization effects on the biological behaviors of cervical cancer cells.The Details are as following.Part1 The correlation study of TAM type and expression of Tim-3 in cervical cancer and their correlation with pathological features[Objective]The relationship between TAM or Tim-3 with cervical cancer staging,infiltration of lymph vessel clearance,lymph node metastasis,living time and the prognosis of patients.[Methods]Cancer tissue and Paracancerous tissue samples are collected from cervical cancer patients respectively.The density and expression of CD68+M1-TAM,CD163+M2-TAM and Tim-3 are detected by immunohistochemical methods.Analysis the relationship of their density and expression levels with the features of cervical cancer.[Results](1)The distribution density of Ml and M2 macrophages in the tissue samples of patients without lymphatic vascular space infiltration was6.9±1.2 and 17.2±1.5,and the expression level of Tim-3 was 0.009±0.001.The distribution density of M1 and M2 macrophages in the tissue specimens with lymphatic vascular space infiltration was 10.6±2.1 and 25.6±3.1,and the expression level of Tim-3 was 0.019±0.003.The expression level of M1 and M2 macrophages in the positive patients was significantly higher,and the difference was statistically significant(P=0.016/P=0.022<0.05).The expression level of Tim-3 was significantly higher,and the difference has statistics meaning(P=0.018<0.05).The distribution density of M1 and M2 macrophage tissue samples in patients with the lymphatic metastasis was 6.6±0.8 and 9.6± 1.3,the expression level of Tim-3 was 0.017±0.004,the incidence of lymph node metastasis of M1 and M2 macrophages in patients with tissue distribution density were 9.8±2.1 and 15.8± 1.9,the expression level of Tim-3 was 0.025±0.003,the expression level of M1 and M2 macrophage positive patients in the sample were significantly higher,the difference was statistically significant(P=0.021/P=0.036<0.05),the expression level of Tim-3 was significantly increased,the difference has statistics meaning(P=0.040<0.05).No distribution density of M1/M2 macrophages infiltration in the tissue samples of patients with uterine were 15.1±2.2/10.3±2.5,the expression level of Tim-3 was 0.009±0.001,distribution density M1/M2 infiltration of macrophages in patients with tissue specimens in uterine were 21.8± 1.3/21.8± 1.7,the expression level of Tim-3 was 0.013±0.002,the expression level of M1 M2 macrophage positive patients in the sample were significantly higher,the difference was statistically significant(P=0.019/P=0.019<0.05),the expression level of Tim-3 was significantly increased,the difference has statistics meaning(P=0.026<0.05).(2)The distribution density of Ml-TAM,M2-TAM in normal tissue near the tumor is(3.92±2.6)/HP and(2.3±1.6)/HP.The distribution density of M1-TAM,M2-TAM in early cancerous tissue is(18.6±2.2)/HP and(5.2±1.1)/HP.The distribution density of Ml-TAM,M2-TAM in advanced tissue near the tumor is(10.6±2.8)/HP and(32.7±4.3)/HP.The Tim-3 protein existed in normal tissues,the early cancerous tissues and the advanced tissues.[Conclusion]The expression levels of Ml-TAM in advanced cervical cancer lower than the early-stage cervical cancer,whereas M2-TAM levels express significantly higher.M1,M2-TAM imbalance may be an important factor in development of cervical cancer.In addition,Tim-3 expression levels were significantly higher in patients of advanced cervical cancer with early stage cervical cancer.The Tim-3 expression can also be judged an important indicator of cervical cancer development.Part 2 The research of cervical cancer about the microenvironment regulation TAM polarization and the expression Tim-3[Objective]Revealed regulation between cervical tumor tissue microenvironment and TAM Polarization or Tim-3 expression.It is expected to provide new methods and new ideas for diagnosis and treatment of cervical cancer in the future.[Methods]Cervical cancer cells were incubated together with Marrow-derived mononuclear cells in vitro.By using the method enzyme-linked immunosorbent assay(ELISA)and RT-PCR,the levels of IL-6,iNOS,MCP-1,IL-10,Arg-1 and Tim-3 were detected.[Results](1)After incubated Marrow-derived mononuclear cells together with the cells from early stage cervical carcinoma,the expression levels of M1-TAM related IL-6,iNOS,and MCP-1 increased obviously(has statistics meaning,P values all less than 0.05).The expression levels of M2-TAM related IL-10 and Arg-1,have no obviously increase(no statistics meaning,P values all more than 0.05).After incubated Marrow-derived mononuclear cells together with the cells from advanced stage cervical carcinoma,the expression levels of M1-TAM related IL-6,iNOS,and MCP-1 have no obviously increase(no statistics meaning,P values all more than 0.05),and the expression levels of M2-TAM related IL-10 and Arg-1 increased obviously(have statistics meaning,P values all less than 0.05).(2)After incubated Marrow-derived mononuclear cells together with the cells from early stage cervical carcinoma,the expression levels of Tim-3 has no obviously increase(no statistics meaning,P>0.05).After incubated Marrow-derived mononuclear cells together with the cells from advanced stage cervical carcinoma,the expression levels of Tim-3 increased obviously(has statistics meaning,P<0.05)[Conclusion]Early stage cervical carcinoma cells could inducted marrow-derived macrophage cells differentiating into M1-TAM.Advanced stage cervical carcinoma cells could induct marrow-derived macrophage cells differentiating into M2-TAM.After differentiating,the Tim-3 expression levels in M2-TAM are higher than M1-TAM.Part 3 The influence of TAM Polarization and Tim-3 expression levels on the proliferation and migration of cervical carcinoma cells[Objective]To reveal the influence of TAM polarization and Tim-3 expression in the proliferation and migration of cervical carcinoma cells.[Methods]Design the siRNA sequence of Tim-3,and then transfect the HeLa cells in vivo.Detect the transfection effect of siRNA by Western blotting method.Detect the change of expression levles E-cadherin and N-cadherin before and after the process of transfection.Detect the migration capabilities of cells by Transwell experiment.[Results]Tim-3 silencing inhibits the migration of cervical cancer cells.Tim-3 in cervical cancer tissue protein promotes cervical cancer cell migration.After silencing Tim-3 by siRNA,the expression of E-cadherin can be promoted significantly,and the expression of N-cadherin can be inhibited,and then inhibit the proliferation and migration of HeLa cells of cervical carcinoma.[Conclusion]Tim-3 can promote the proliferation of cervical cancer.After silenced the expression of Tim-3,the rate of cervical cancer cell proliferation can be reduces.The expression of Tim-3 plays an enhanced role in the invasive ability of cervical cancer tissue cells. |
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