| Tumor expansion not only rests with tumor cell itself, but also is closely related with different factors in internal environment. As early as 1889 Stephen Parget proposed the hypothesis of"seed and soil"and used to describe the relation of the tumor and microenvironment. The mammary cancer microenvironment is the internal environment composing of the active medium, which mammary cancer partial infiltrated immune cell, the interstitial cell and secretion and so on, and the mammary gland cancer cell. Mammary cancer and its micro- environment not only depends on each other mutually, promotes mutually, but also oppresses mutually, struggles mutually. To a certain extent it will delay the tumor expansion and enhance patient's lifetime by changing certain factors in the microenvironment.At present, the top clinical treatment of mammary cancer is still with surgical operation, chemotherapy, radiotherapy and endocrine therapy. These traditional curable methods can rise very nice curable effect to some sufferers of mammary cancer on early or medium metaphase. But they aren't capable of controlling pathological changes well to the mammary cancer sufferers on later period with long- distance metastasis. Along with the immunology development, the tumor immunity treatment has obtained the remarkable treatment effect in vitro research and the animal experimentation, but the clinical practice is only restricted in phase I, II of clinical trial at present and the curable effect is unsatisfactory. The above situation is closely related with the complex immunity regulating network in microenvironment. And the immunity endures in microenvironment is the most important factor devoting to the above situation. There are many mechanisms to induce immunotolerance in the microenvironment, but the microenvironment internal inhibition germ cell and some suppress the germ cell factor play the most important roles.Regulatory T cells (Treg), an important inhibitory cell, can inhibit lymphocyte function in tumor microenvironment by infiltration ways and keep antigen special immunotolerance. The research indicates that Treg has been discovered in the lung cancer, mammary cancer, stomach cancer, colon cancer and so on many kinds of common human body tumors, and the tumor partial T cell function to come under obvious suppression has been observed simultaneously. Some researchers discovered that it could reverse immunotolerance and enhance the tumor immunity treatment effect by suppressing Treg immunotolerance function. People believed that Treg was CD4~+CD25~+Treg by initial research. But with the development, people discovered that the Foxp3~+ expressed in the Treg cell nucleus particularly, so believed that Foxp3~+ was the key factor during Treg growth, activation and display function. Therefore examining expression level of CD4~+CD25~+Foxp3~+ can reflect Treg level really. The transforming growth factor-beta (TGF-β) is the most effective inhibiting factor inducing tumor immunity to escape, which can widely suppress the tumor patient's immune system to cause the tumor cell escape organism immunity surveillance function and multiplies rapidly. The research indicates TGF-βat prostate gland cancer, stomach cancer and so on the malignant tumors assumes expresses excessively, which may express by the tumor cell or many kinds of matrix cells. The expression excessively may cause the tumor to transform and progress, even may also decide the patient prognosis. The interleukin-10 (IL-10) is another important inhibiting cell factor playing significant role in tumor immunity. IL-10 plays its role by inhibiting the function of antigen presenting cell and T lymphocyte.In the past, people took it for granted that it was unfit for immunotherapy followed by chemotherapy and radiotherapy. But the view that special chemotherapy and radiotherapy can improve curable effect by changing the body inhibitory prevails now. Dendritic cells (DCs) having formidable T cell activation ability are the most formidable antigen presenting cell discovered at present.They can obviously stimulate the initial T cell, B lymphocyte, NK cell's multiplication and activeness. Therefore, DCs as organism immune response initiator play an important role in the immune response, and tumor vaccine based on DCs holds the key position in the tumor immunity treatment.In this study we observe expression level of CD4~+CD25~+Foxp3~+Treg, TGF-β, IL-10 in BALB/c mammary cancer model made by injecting 4T1 mammary cancer cells, then select the optimal interfering combination and time by special chemotherapy and radiotherapy to mammary cancer. Based on the above experiment, we study curable effect by inoculating dendritic cells (DCs) vaccine to explore the role of immunotolerance in mammary cancer microenvironment during tumor produces, expansion and metastasis and provide a basis for chemo-radiotherapy and DCs therapy.Part 1 Expression and its role of CD4~+CD25~+Foxp3~+Treg, TGF-βand IL-10 in mammary cancer microenvironmentObjective: To investigate the role and mechanism of CD4~+CD25~+Foxp3~+Treg in immunosuppression of mammary cancer.Methods: There were 40 mammary cancer samples (20 ones with lymphonodus metastasis, 20 ones without lymphonodus metastasis) and 20 mammary fibroma ones selected from General Surgery of ours. Expression level of CD4~+ CD25~+ Foxp3~+Treg, TGF-βand IL-10 in original, peripheral tissue, axilla lymphonodus and mammary fibroma of the above samples were measured respectively by FACS and immunohistochemical method.Results: The expression level of CD4~+ CD25~+ Foxp3~+Treg, TGF-βand IL-10 in original tissues of mammary cancer were all higher than that of mammary fibroma (P<0.05), and were higher than that of peripheral tissue and axilla lymphonodus. In the group with lymphonodus metastasis, the expression level of the above three biological measured makers in mammary cancer peripheral tissue was significantly lower than that expressed in axilla lymphonodus (P<0.05), while in the group without lymphonodus metastasis, there wasn't any difference in expression level of the above three measured index between mammary cancer peripheral tissue and axilla lymphonodus (P>0.05). Part 2 Study of the effect of special chemotherapy and radiotherapy on CD4~+CD25~+Foxp3~+Treg, TGF-βand IL-10 in BALB/c in mammary cancer microenvironmentObjective: To observe effect on CD4~+CD25~+Foxp3~+Treg, TGF-βand IL-10 in BALB/c mammary cancer microenvironment by special chemotherapy and radiotherapy, and to select the optimal interfering group.Methods: There were 640 BALB/c mice divided into four groups at random, named as control, chemotherapy, radiotherapy, and chemotherapy combined with radiotherapy group respectively. Subsequently BALB/c mice were subcutaneously inoculated 4T1 cells as tumor model and were cured after 16 days. Expression level of CD4~+CD25~+Foxp3~+Treg measured by FCM method and TGF-β, IL-10 measured by immunohistochemical method were respectively analyzed at the 18th, 20th, 22nd, 24th day after inoculating vaccine.Results:①CD4~+CD25~+Foxp3~+ Treg expression level of chemotherapy group at the 20th day, radiotherapy group at the 18th day, chemotherapy combined with radiotherapy group at the 18th and 20th day were all lower than that of control group (P<0.05). And the expression level of chemotherapy combined with radiotherapy group at the 18th and 20th day were lower than that of chemotherapy group and radiotherapy group at the 18th and 20th day (P<0.05).②There wasn't statistical significance between chemotherapy group and control group in expression level of TGF-βand IL-10(P>0.05), while that of radiotherapy group at the 18th and 20th day after inoculating vaccine was lower respectively than that of control group (P<0.05).And expression level of TGF-βand IL-10 in the chemotherapy combined with radiotherapy group at the 18th and 20th day was respectively lower than that of control group and chemotherapy group (P<0.05), but there wasn't statistical significance comparing with that of radiotherapy group(P>0.05).Part 3 Study of the effect of immunotherapy based on interfering mammary cancer microenvironmentObjective: To observe immunotherapy effect based on chemotherapy combined with radiotherapy interference in BALB/c mammary cancer microenvironment.Methods: There were 640 BALB/c mice divided into four groups at random, named as control, chemotherapy combined with radiotherapy group, DCs vaccine group, chemotherapy combined with radiotherapy and DCs vaccine group respectively. Subsequently BALB/c mice were subcutaneously inoculated 4T1 cells as tumor model and started to be cured after 16 days. Expression level of CD4~+CD25~+Foxp3~+Treg measured by FCM method and TGF-β, IL-10 measured by immunohistochemical method were respectively analyzed at the 20th, 22nd ,24th and 26th day after inoculating vaccine.Results:①CD4~+CD25~+Foxp3~+Treg expression level of chemotherapy combined with radiotherapy group at the 20th day was lower than that of control group (P<0.05). The expression level of DCs vaccine group at the 22nd, 24th and 26th day were respectively lower than that of control group and chemotherapy combined with radiotherapy group(P<0.05).And the expression level in chemotherapy combined with radiotherapy and DCs vaccine group at the 20th,22nd, 24th and 26th day were respectively lower than that of the other three groups(P<0.05).②The expression level of TGF-βand IL-10 in chemotherapy combined with radiotherapy group at the 18th, 20th day were lower than that of control group(P<0.05).The expression level in DCs vaccine group at the 22nd ,24th and 26th day were lower than that of control group and chemotherapy combined with radiotherapy group(P<0.05). And the expression level in chemotherapy combined with radiotherapy and DCs vaccine group at the 20nd, 22th ,24th and 26th day were all lower than that of the other three groups(P<0.05).Conclusion:①The examination on CD4, CD25 and Foxp3 three mark streaming cytology can more accurately reflect Treg level in mammary cancer organizes.②With the expansion of tumor, expression level of CD4~+CD25~+Foxp3~+Treg, TGF-βand IL-10 are rising gradually.③CD4~+CD25~+Foxp3~+Treg,TGF-βand IL-10 play an important role in immunotolerance in mammary cancer microenvironment.④Treg in mammary cancer microenvironment has immunotolerance function by secreting inhibitory factor such as TGF-β, IL-10, simultaneously TGF-βand IL-10 can promote CD4~+CD25~+Foxp3~+Treg secretion. There is some synergistic action between CD4~+CD25~+Foxp3~+Treg and TGF-β, IL-10.⑤It is the 4th day after applying drugs that single CTX (100mg/kg) unites single Gamma beam (2Gy) local irradiation affects mammary microenvironment most obviously. The radiotherapy combined with chemotherapy treatment surpasses pure chemotherapy and pure radio- therapy obviously regarding to mammary cancer microenvironment influence.⑥Low dose chemotherapy and radio- therapy doesn't have antitumor effect, but has an modulated effect on mammary microenvironment.⑦The expression level of CD4~+CD25~+Foxp3~+ Treg, TGF-βand IL-10 can fall remarkably by pure DCs vaccine treatment, which indicates DCs can activate the activity of the body effect T cell and strengthen body anti- tumor immunity capability.⑧The curable effect by inoculating DCs vaccine based on special chemo-radiotherapy is better than that by pure DCs vaccine treatment.⑨The expression level of CD4~+CD25~+Foxp3~+Treg, TGF-βand IL-10 in mammary cancer micro- environment all falls by chemotherapy, radiotherapy and DCs vaccine treatment, which indicates that these treatment methods can inhibit the number and function of Treg and some inhibitor factors such as TGF-βand IL-10.Thus it inhibits immunotolerance occurrence in tumor micro- environment and improves curable effect.
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