Experimental Research On The Effect Of Human IL-8 Gene On Invasive And Angiogenic Ability Of Cervical Cancer

Objective: Uterine cervix carcinoma is the second commonest female cancer worldwide, The Prevalence lies in first Place in our country (China) [1].The areas of its high occurrence are all connected with each other and people who developed it are at a much younger age than before[2]. It has become one of the main diseases which severely threaten the health of women in our country. The occurrence of cervical cancer development is a quantitative change to qualitative change, from gradient to the mutation process. Local infiltration and lymph node metastasis not only influence the operation effect but also correlate directly with the prognosis of patients. Lymph node metastasis is the main route of metastasis of uterine cervix carcinoma, and could take place in the early stage. It has been reported that the 5-year survival rate of the patients who had no lymph node metastases was 82.2% and who had lymph node metastasis was 50.8%[3].Angiogenesis is the process that from the previously blood capillary system to form new vessels, and plays important part in the physiology and pathology process. The regulation of angiogenesis depends on the balance between angiogenesis factors and vascular static factor factors, they discern to promote and restrain the tumor's angiogenesis. If there is no blood vessel, the diffusion of oxygen and nutrient substance will be restrained, then, the tumor can not grow up. Angiogenesis process has many growth factors'trigger, These growth factors are from tumor cell-self or their peripheral matrix cell[4]. Along with the last years'investigation of angiogenesis,people discovered that all manners of growth factors are all can promote angiogenesis. Angiogenesis correction chemotatic factors and cytokine play important part in angiogenesis process, among these factors, IL-8 which has been described neutrophilia chemical inductor initial,is one of CXC chemotatic factors, today, more and more people pay attention to it.IL-8 was discovered in 1987, the first one, etc. Yoshimur[5] chemotactic factor, was then named monocyte-derived neutrophil chemotactic factor is associated with inflammation-related CXC chemokines. Along with the investigation functional mechanism of IL-8 and its acceptor, Strieter[6] discovered that NH2 termination of IL-8 had a ELR motif, playing an important role in angiogenesis. Murphy[7] also found that IL-8 involved in tumor angiogenesis, and tumor invasion, metastasis. Followed by researchers in small cell lung cancer[8] and stomach cancer[9] and ovarian cancer[10], and so have come to similar results, suggesting that IL-8 involved in tumor occurrence and development. IL-8 can promote the proliferation of tumor cells to tumor cells to endothelial cell [11] migration; regulating tumor cells and surrounding stromal cells secrete MMP-2, MMP-9 and to enhance its activity, thereby contributing to collagenase activity and increased in vitro invasion[12] of tumor cells. IL-8 can also promote vascular endothelial cell proliferation, migration, inhibit the spontaneous apoptosis and increased permeability of blood vessels[13] contribute to tumor cell metastasis. In addition, studies have shown that lymphatic endothelial cells through the secretion of chemokines in attracting tumor cells, and then take the initiative to promote lymph node metastasis of tumor cells. But until now, there has been little investigation of IL-8 in uterine cervix cancer. Tjiong[14] and others by comparing the cervical secretions and vaginal washing fluid levels of IL-8 that the cases of cervical cancer and CIN may have high levels of local vaginal IL-8 and IL-6, these cytokines may have local inflammatory response and tumor growth of a role. Japan's Fujimot [15] and others studies have shown that IL-8 and cervical cancer angiogenesis and prognosis, but did not clarify the molecular mechanism of action. Suhui Wu[16], etc. carried out in early cervical squamous cell carcinoma cDNA microarray screening results show that IL-8 in a lymph node metastasis of cervical cancer tissue than those without lymph node metastasis was 3.4 times higher, suggesting it may at an early stage of cervical cancer invasion and metastasis play an important role. Although the research on the treatment of UCC has continued for nearly a hundred years and the result is very striking, the 5-year survival rate of early UCC hasn't reached 90%-100%. To improve the treatment and promote the curative effect,in recent years, with the technological development of cell engineering and generic engineering, tumor immunotherapy is also developing rapidly. Recently cytokine gene therapy has become the focus of research. In this study, through animal experiments, immunohistochemical staining compared CD34 and CD57 in cervical tumor tissue expression was observed IL-8 in cervical cancer invasion and metastasis ability of and explore their mechanisms of action, with a view to cervical cancer The treatment and prognosis of new indicators. Now more than immunohistochemical techniques for quantitative microvessel count, that microvessel density count, this is MVD. Microvessel density, reflecting the markers are CD34, CD31, CD105, and vs factors, among which CD34 is a more sensitive and more specific for tumor angiogenesis endothelial cell surface marker, CD34 known as hematopoietic stem cell antigen targeted Wei Ren, in the hematopoietic stem cells, vascular endothelial cells, and others have expressed. In tumor tissues, its expression and tumor vascular endothelial cell proliferation, migration and related, and is considered to determine the prognosis of cancer patients as an independent factor. NK cell is one effector cell which has spontaneous cytototix reactive and effects on many kinds of target cells on immune function of organism. It play in the main anti-tumor non-specific immune function,It is neither need a complex antigen-presenting role nor restriction from the major histocompatibility complex (MHC), to release specific anti-protein -- pertorin (pertorin), cytotoxic factor (NKCF ) directly. It can dissolute and kill the target cell, so it is considered to be the body of anti-tumor, anti-infection of the first line of defense[17,18]. CD 57 (I-INK 1 1, leu-7) is a glycoprotein with a molecular weight of 110000, is considered the specificity of human natural killer cell surface markers. Also present in T lymphocytes and some tumor cell surface [19,20].The formerly study indicated that CD57 antigen can hold back the proliferation of homeocyte . In peripheral blood of 30% -80% of the NK cells can express views antigens, so the level of positive cells to some extent reflect the function of NK cells.Methods:1 Cells and experimental animalHela/pcDNA3.1(+)–h/IL-8 cell and Hela/pcDNA3.1(+)–h cell , these cells all come from laboratory.Nude-mice, 21, female, aged 4-6 week, body mass is 16-18g, buy from animal Center of Beijing Concord.2 Hela/pcDNA3.1 (+) -h/IL-8 cells, Hela/pcDNA3.1 (+)-h cells and Hela cells were cultured to logarithmic growth phase, taking 1.5×107 cells (0.2ml), conventional After skin disinfection and inoculated subcutaneously in nude mice, after the right, when the tumor grew to a diameter of about 5 mm were regarded as tumorigenicity, and record the animal tumorigenicity time and rate.3 After animal tumorigenicity, to measure every group tumor's length and width, through formula V=π/6×a×b2 to calculate tumor volume(mm3).4 9 weeks after inoculation of tumor cells mice were sacrificed cervical dislocation, complete stripping tumor tissue and lung tissue.5 The nude mice tumor was paraffin-embedded and prepared into histologic section, and the tumor cells morphology was observed by HE staining, the expression of CD34 and CD57 in tumor tissue was detected by immunohistochemical staining.Result:1.The subcutaneously transplant nude mouse models were successfully established. The animal tumorigenicity rate are all 100%. Hela/pcDNA3.1(+)–h/IL-8 cell group's tumorigenicity time is obviously lower than Hela/pcDNA3.1(+)–h cell group's and Hela cell group's(P<0.05),there are no obviously distinction between Hela/pcDNA3.1(+)–h cell group and Hela cell group(P>0.05).2.The gross tumor volume of the 7,8,9week,the Hela/pcDNA3.1(+)–h/IL-8 cell group'volume obviously bigger than Hela/pcDNA3.1(+)–h cell group and Hela cell group(P<0.05). After subcutaneously transplant,before sixth week the volume comparion has no significant differenct (P>0.05).3.The immunohistochemical resultThe immunohistochemical result of MVD: The CD34 expression of Hela/pcDNA3.1(+)–h/IL-8 cell group is obviously.Hela/pcDNA3.1(+)–h/IL-8 cell group obviously higher than Hela/pcDNA3.1(+)–h cell group and Hela cell group(P<0.05), there are no obviously distinction between Hela/pcDNA3.1(+)–h cell group and Hela cell group(P>0.05).The immunohistochemical result of CD57: Hela/pcDNA3.1(+)–h/IL-8 cell group obviously less than Hela/pcDNA3.1(+)–h cell group and Hela cell group(P<0.05). there are no obviously distinction between Hela/pcDNA3.1(+)–h cell group and Hela cell group(P>0.05).4.Lung tissue of each group have no cancer metastasis.Conclusion:1.IL-8 gene was successfully constructed in nude mice transplant model, by comparing each group into a tumorigenicity rate , time and tumor volume confirmed IL-8 can promote tumor growth.2.Animal study found that IL-8 can promote the generation of capillaries within the tumor tissue,and this provides the necessary conditions for the growth of cervical carcinoma invasion and distant metastasis .3.Low expression of CD57 may be related to the growth of cervical carcinoma invasion and distant metastasis.4.Lung tissue of each group have no cancer metastasis.