| Digestive system from the digestive tract and digestive gland of two parts. Digestive tract including the mouth, pharynx, esophagus, stomach, small intestine(duodenum, jejunum, ileum) and large intestine(cecum, appendix, colon, rectum, anal canal) and other units. Clinically often put the mouth to the duodenum, said this section of the digestive tract, the following part of the jejunum, said the lower digestive tract. There are small and large digestive gland gland gland two kinds. Scattered in small gland in the digestive tract wall ministries, large digestive gland has three pairs of salivary glands(parotid, submandibular gland, sublingual gland), liver and pancreas. Human digestive system is one of the nine systems. Oral is the initial part of the digestive tract. An excuse to crack with the outside world before, after continued with angina and pharynx. Oral cancer is the most common malignant tumors, the incidence of systemic cancer ranks sixth, local recurrence rate and poor prognosis, more than half of advanced oral cancer patients survive less than one year. More than 90% of oral cancer is oral squamous cell carcinoma, can be a primary in the mouth, but also by distant metastasis to bone tissue or adjacent structures, or the primary in violation of adjacent tissue lesions in the mouth and Performance oral tissues. Oral squamous cell carcinoma risk factors varied, including physical carcinogens, chemical carcinogens, biological carcinogens, genetic, organism susceptibility and ethnicity. Studies have shown that a single factor does not lead to oral squamous cell carcinoma. Oral squamous cell carcinoma is most likely the combined effects of internal and external factors in a long time. Oral squamous cell carcinoma are more prone to neck lymph node metastasis, the rate was 16%-55%, as long as there is a single ipsilateral or contralateral cervical lymph node metastasis, survival rate that is reduced to 50%. Therefore, whether oral squamous cell carcinoma with lymph node metastasis in patients for prognosis and treatment programs to determine the judgment has very important significance.Cancer invasion and metastasis of oral squamous cell carcinoma led to poor prognosis, one of the main recurrence rate. Malignancy and fixed parts of the anatomy of blood, lymphatic drainage is difficult to fully explain the cancer invasion and metastasis. Understanding of cancer metastasis modern medicine can be summarized as the following aspects: First, the heterogeneity of tumor biological characteristics. Secondly, metastasis of malignant tumor is a highly complex process selective to provide a suitable tumor cell subpopulation invasive growth phenotype within a stable environment. Also, the final result of the transfer of malignant tumor invasion generated depends on the interaction with the environment within the cell, tumor cell capture by the environment within the resources to get the advantage of the growth.A key hallmark of cancer cells is to evade anoikis or detachment-induced apoptosis. By allowing cancer cells to survive in inappropriate places and to disseminate through the body, anoikis resistance is a critical determinant of tumor progression and metastasis and represents as a critical barrier to cancer treatment. Hence, one important therapeutic strategy in curtailing tumor aggressiveness and metastasis is to restore or induce anoikis sensitivity in cancer cells.Malignant cells in the cell matrix is usually caused by the motion and moving cycle of four steps, namely the cells extending pseudopodia formation of the head and outward to establish a new site of adhesion, and the cell body shrinkage tail withdrawal, through continuous migration of the surrounding repeated four round processes, the precise molecular mechanisms of regulation of this process is very complex, involving multiple intracellular signal transduction pathways.Bit-1 was found as an open reading frame when exploring the integrin-mediated regulation of Bcl-2 expression in 2004.The protein was named as bit-1(bcl-2 inhibitor of transcription 1) to denote its ability to regulate Bcl-2 expression. Present study suggests that Bit-l is an important by the integrin-induced pro-apoptotic factors, mediated by cell adhesion induced de-nest apoptosis. Integrins(integrin)-mediated cell adhesion is a major regulator of apoptosis nest one of the factors. Degranulation induced by the adhesion of the nest has been confirmed in a variety of apoptotic cell types, such as endothelial cells, fibroblasts, neuronal cells, intestinal epithelial cells, kidney cells and prostate epithelial cells. The study found that integrin receptor protein can regulate apoptosis of epithelial cells, epithelial cells sustained by getting suspended, or closed with a short peptide to disrupt the interaction between epithelial cells and the ECM can induce apoptosis. Further studies showed that integrin receptor protein via conduction through the induced signal to the cells in contact with the ECM, to inhibit cell apoptosis.Later results showed that it was evolutionarily conserved from bacteria to human. The Bit-1 transcript was prominent in human testis, prostate, skeletal muscle and liver, and was clearly detectable in human heart, spleen, placenta and colon. No significant Bit-1 m RNA sigal was seen in the thymus or in peripheral leukocytes.Bit-1 is a 179-residue mitochondrial protein which appears to be part of a previously unknown apoptosis pathway that is regulated by integrin-mediated cell attachment. As an anoikis effector, Bit-1 is released from mitochondria in cells cultured in suspension and forms a complex with the transcriptional regulator protein. Amino-terminal Enhancer of Split(AES). The formation of the Bit-1-AES complex initiates a caspase-independent form of apoptosis. Integrin-mediated cell adhesion appears to be the only upstream anti-apoptotic treatment that can rescue cells from apoptosis induced by Bit-1. Knowledge of the molecular workings and significance of the Bit-1 apoptotic pathway may help design new ways of initiating anoikis for therapeutic purposes. Cytoplasm containing higher Bit-l concentration, the more significant may promote cell apoptosis, which contain low concentrations of Bit-1 can reduce the occurrence of apoptosis. That is, Bit-l protein and cell apoptosis is a very close new molecules, therefore, has decided to study this mitochondrial apoptosis proteins for exploring and regulation of apoptosis of cancer cells have important clinical significance.The purpose of this study was to investigate the distribution and biological effects of Bit-1 protein in human oral squamous cell carcinoma of Tca8113 and to explore the mechanism of action, in order to provide some new ideas for the treatment of oral squamous cell carcinoma.Our research can be divided into three parts as following:(?)Expression of Bit-1 m RNA and protein in oral squamous cell carcinoma and its Clinical Significance.Methods: To explore the relationship between Bit-l with oral squamous cell carcinoma, the study by reverse transcription polymerase chain reaction(reverse trancriptase-polymerase chain reaction, RT-PCR) and immunohistochemistry(Immunohistochemistry) for oral squamous cell carcinoma, atypical hyperplasia adjacent tissues and normal oral mucosa Bit-l expression levels were detected, explore the relationship between Bit-I gene and protein expression and oral squamous cell carcinoma. 1. Semi-quantitative RT-PCR method to detect 75 cases of oral squamous cell carcinoma(25 cases with lymph node metastasis) and paired adjacent tissues, Bit-l m RNA expression in normal oral mucosa. 2. Detected 75 cases of oral squamous cell carcinoma, 25 patients with lymph node metastasis by immunohistochemical SP method expression next 25 cases and 25 cases of normal tissues tissue specimens Bit-l protein.Results: 1 Bit-I m RNA expression in oral squamous cell carcinoma Bit-l m RNA carcinoma, adjacent atypical hyperplasia and normal oral squamous cell carcinoma of the content of the order of 0.879±0.136,0.514 ± 0.093,0.438±0.071. Bit-l m RNA expression levels in oral squamous cell carcinoma and dysplasia were higher than normal oral mucosa( all P <0.05), whereas itsexpression in atypical hyperplasia and normal oral mucosa were no significant difference. 2 Relationship Bit-l m RNA expression and clinicopathological parameters Statistical results showed that oral squamous cell carcinoma,the expression level of the patient's age Bit-1, sex was no significant correlation(P> 0.05), but the expression of Bit-l genes in lymph node metastasis group was significantly higher at no lymph node metastasis group was statistically significant between the two groups. TNM staging of expression but also in stage III / IV group was significantly higher than 1 / 11 group and was statistically significant(P <0.05). 3 Bit-l protein expression in the oral mucosa By immunohistochemistry assay Bit-1 in oral squamous cell carcinoma primary tumor and normal oral mucosa expression. Bit-1 is mainly expressed in the cytoplasm and cell membrane of tumor cells in normal oral mucosa showed low staining or staining. Our experimental results show that : in 75 cases of oral squamous cell carcinoma primary tumors positive in 54( 72%), while in 25 cases of normal oral mucosa expression only 2 cases( 8%), statistical analysis showed Bit-1 significant difference(p <0.05) in the expression between the two groups. 4 Relationship Bit-l protein expression and clinicopathological parameters Pathological diagnosis combined specimens, HE staining and surgical records for all specimens in accordance with the degree of differentiation, gender, age, TNM stage and lymph node metastasis classification, and statistical analysis showed that : in stage ?- ?oral squamous cell carcinoma primary tumor Bit-1 expression in 24 patients( 24 /40), and ?- ?oral squamous cell carcinoma primary tumor Bit-1 expression in 30 cases( 30/ 35), significantly higher than the former expression, both groups were statistically significant. Lymph node metastasis in oral squamous cell carcinoma Bit-1 expression in 21 cases(21/ 25), without lymph node metastasis in oral squamous cell carcinoma Bit-1 expression in 33 cases(33 /50) expression with lymph node metastasis of oral squamous cell carcinoma bit-1 expression wassignificantly higher than that associated with lymph node metastasis, and between the two groups was statistically significant(P <0.05). Statistical analysis showed that: Bit-1 expression in oral squamous cell carcinoma with TNM stage and lymph node metastasis(P <0.05), whereas no significant relationship with sex, age, tumor size, degree of differentiation of patients(P> 0.05)(?)The role of Bit-1 in the invasion and migration in oral squamous cell carcinomaMethods: First, we constructed the eukaryotic expression vectors p CMV-Myc-bit-1 and p CMV-Myc-EGFP. The expression of endogenous bit-1 in oral squamous cell carcinoma cell was detected by RT-PCR and Western Blot before transfection. Then we determined the optimal transfection condition using expression vector p CMV-Myc-EGFP, and transfected oral squamous cell carcinoma cell Tca 8113 with the vector p CMV--Myc-bit-1 which containing the human gene bit-1 under the optimal condition. After the transfection, we detected the bit-1 expression level of the transfected cells. MTT method was used to determine the activity of the transfected cells. Application of Transwell migration assay chamber before and after transfection Tca 8113 cells.Results: 1. RT-PCR and Western Blot showed that the expression of a small amount of human squamous cell carcinoma of the target gene Tca 8113 only. And after Tca 8113 cells transfected with p CMV-Myc-bit-l eukaryotic expression vector, RT-PCR and Western Blot showed that transfected cells have a more clear Bit-1 gene expression. 2. MTT assay results showed that untransfected oral squamous cell carcinoma Tca8113 and p CMV-Myc transfected with oral squamous cell carcinoma Tca8113 culture during the experiment, as the culture has been a long time, the number of cell survival continue to increase, no significant difference between the two. The p CMV-Myc-bit-l transfected with oral squamous cell carcinoma Tca8113 the cultureduring the experiment, as time has been long, the number of survival cells decreased. 3. Cell invasion assay Transwell invasion assay confirmed, p CMV-Myc-bit-l transfected with oral squamous cell carcinoma Tca8113 after reducing its invasiveness. Photographed under a microscope and counting the number of randomly selected ten different ability p CMV-Myc-bit-l transfected cells through the basement membrane than p CMV-Myc-transfected cells and cells transfected with the difference, the number of films were wearing : 78.43 ± 12.64,69.35 ± 11.38,35.71 ± 6.92.(?)The role of Bit-1 in the proliferation and apoptosis in oral squamous cell carcinomaMethod: 1 Acridine orange experiments Prepare transfected cells were transfected after 24 h, 48 h and 72 h depicting a hole non-transfected cells, p CMV-Myc-transfected cells and p CMV-Myc-bit-l-transfected cells, single cell suspension was prepared. Take 95 ul cell suspension, adding 5ul acridine orange stock solution(0.lml/mg, dissolved in PBS), after mixing the droplets absorb a drop mixed on a glass slide, coverslip, fluorescence microscopy and photographed. 2 DNA fragment analysis experiments 48 h after transfection cells were collected from non-transfected, p CMV-Myc-transfected cells and p CMV-Myc-Bit- transfected cells. Positive control cells lyophilized kit provided U937 cells grounds. The method according to apoptotic DNA Ladder kit instructions and steps to extract DNA. 1% agarose gel electrophoresis and photographed under UV irradiation apparatus. 3, Flow cytometry experiments Rate of apoptosis by flow cytometry bit-l gene transfection of human oralsquamous cell carcinoma Tca 8113 and the cell cycle. Respectively, after transfection 24 h, 48 h and 72 h to collect non-transfected cells, p CMV-Myc-transfected cells and p CMV-Myc-bit-l-transfected cells, FCM analysis of apoptosis and cell cycle disorder cases. 4 PI single staining to detect cell cycle Untransfected cells, the p CMV-Myc-transfected cells and cells transfected with DNA p CMV-Myc-bit-l propidium iodide stained cells by flow cytometry for DNA content and DNA content in accordance with distribution, performed cell cycle and apoptosis analysis.Results: The results of acridine orange staining, DNA Ladder and FACS also showed that as the culture time went, the apoptosis rate increase in the cell strain Tca 8113 transfected by p CMV-Myc-bit-1, compared with control group, the untransfected cells and cells transfected by p CMV-Myc. The apoptotic cells in the control group were very rate during the different culture time.Conclusion In summary, this study should first RT-PCR and Western Blot analysis technique in human oral squamous cell carcinoma in the distribution of human Bit-1 protein, followed by a more comprehensive observed that the protein in human oral squamous cell carcinoma Tca 8113 the transfer value, the role of apoptosis has laid a good foundation for further analysis Bit-1 mechanism of action, and it is possible for the biological treatment of oral squamous cell carcinoma provide a new target molecules. |
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