| Background and objectiveCervical carcinoma is a common malignant tumor in China, one of the female reproductive system cancer incidence is in the first place, and getting younger. The etiology and pathogenesis of cervical carcinoma is not yet very clear, but many studies have shown that its occurrence, development, and invasion and metastasis is a multi-factor, multi-stage, multi-gene involved a complex process, therefore, to seek and cervical cancer occurrence and development of related indicators has become a research hot spot.Midkine (midkine, MK) is found by Kadomatsu such as differences in hybridization methods using retinoic acid-induced mouse embryonic tumor cell line HM-1 cell cDNA library were screened out of a new gene. The initial study found that nerve cells can promote neurite outgrowth, neural stem cell differentiation and embryonic neuronal survival and so on. After the study found that in physiological circumstances MK gene expression change with the development of individual, high expression in the embryonic period, gradually decreased after birth, in the adult kidney and intestinal epithelial tissues other than the part of almost no expression. In the case of the disease, especially malignant tumors in MK mRNA with high intensity, high-frequency expression. The possible occurrence of tumor, growth and metastasis. Studies have shown that MK in gastric cancer, esophageal cancer, pancreatic cancer, osteosarcoma, endometrial cancer, and nervous system tumors and other tissues over-expressed. On MK expression in cervical carcinoma as well as with cervical cancer occurrence and development of the relationship between the study of only one reported in the literature. To further explore the MK expression and cervical cancer occurrence and development of relations; to find suppression of cervical cancer occurrence, development and invasion and metastasis of an effective method, this paper by using immunohistochemistry, in situ hybridization to detect 60 cases of cervical squamous cell carcinoma, 29 cases of cervical intraepithelial in neoplasia (cervical intraepithelial neoplasia, CIN) organizations, and 48 cases of normal cervical tissue MK protein and mRNA expression; MK antisense oligonucleotides by cationic liposome-mediated transfection Hela229 cervical squamous cell carcinoma cells, the use of immune cells chemistry, in situ hybridization to detect cells transfected Hela229 expression of MK protein and mRNA. For the application of MK antisense oligonucleotide targeted therapy to provide a theoretical basis for cervical cancer and other malignancies.Materials and methods1. Materials: 60 cases of cervical squamous cell carcinoma, 29 cases of CIN organizations, and 48 cases of normal cervical tissue for the Pingdingshan Coal Group Co. General Hospital, and the 3 Affiliated Hospital of Zhengzhou University from March 2004 to December 2008 between the surgical resection specimens; human cervical squamous cell carcinoma Hela229 cells from Chinese Academy of Sciences Shanghai Institute of Cell Biology.2. Methods: (1) using immunohistochemistry, in situ hybridization detection of cervical squamous cell carcinoma, CIN and normal cervical tissue organization and MK protein and mRNA expression;(2) Design and Synthesis of 2 MK antisense oligonucleotide (ASODN1, ASODN2) and an unrelated sequence oligonucleotide (N-ODN);(3) were used 150μg/ml, 200μg/ml, 250μg/ml 3 different concentrations of MK ASODN1, ASODN2 and N-ODN in vitro cultured Hela229 grouping cells transfected with 48h and 72h. A separate blank control (without antisense oligonucleotide-liposomes) and cell blank control (without any operation);(4) using immunocytochemical methods and intact cells was observed by in situ hybridization MK ASODN right Hela229 cells MK protein and mRNA expression.(5) the use of SPSS 11.0 statistical software for statistical analysis. Was used to compare between the rate of positive tests x~2; measurement data to mean±standard deviation (±S), said two groups means were compared using two-sample t-test (t-test); for more than two groups were used to be compared with analysis of variance ( ANVOA); toα= 0.05 for the significance level.Results1. MK protein and mRNA expression: MK protein and mRNA in cervical squamous cell carcinoma Expression of CIN exist in the organization, while no expression in normal cervical tissue. Protein-positive staining in cervical squamous cell carcinoma and CIN tissue cell and (or) the cytoplasm, brownish yellow granules; MK mRNA located in cervical squamous cell carcinoma and CIN organizations within the cytoplasm, showing blue-purple granules.2. Immunohistochemistry Results: In normal cervical epithelium, CIN and cervical squamous cell carcinoma of the organization MK protein expression rates were 0.00% (0/ 48), 20.69% (6/29) and 65.00% (39/60 ). the groups using Chi-square test ,the P-value was less than 0.01 Compared between groups using partitions of Chi square methond,the differences were significant (P<0.05).3. In situ hybridization: MK mRNA in normal cervical epithelial tissue expression of negative 0.00% (0/48) in CIN and cervical squamous cell carcinoma of the organization MK protein expression rates were 31.03% (8/29) and 71.67% (43/60). Compared the groups using Chi-square test ,the P-value was less than 0.01 Compared between groups using partitions of Chi square methond,the differences were significant (P<0.05).4. MK ASODN pairs of cervical squamous cell carcinoma Hela229 cells MK protein expression: Hela229 There MK protein expression in cells located in the cytoplasm Hela229, brownish yellow granules. 3 different concentrations of MK ASODN1, ASODN2 right Hela229 cells MK protein expression were significantly inhibited, and in order to 250μg/ml ASODN1 the strongest inhibitory effect (P<0.05), 72h to 48h stronger than the action effect (P<0.05); but no significant difference was found in different concentration and antisense oligodeoxynucleotides (P>0.05) . N-ODN transfected group and the control group and ASODN1, ASODN2 compared with analysis of variance in all experimental groups, the differences were significant (P <0.05 or P<0.01). Multiple comparison among the means was significant(P<0.05). 5. MK ASODN pairs of cervical squamous cell carcinoma Hela229 cells MK mRNA expression: Hela229 There MK mRNA expression in cells located in the cytoplasm Hela229, showing blue-purple granules. 3 different concentrations of MK ASODN1, ASODN2 right Hela229 cells MK mRNA expression also showed a significant inhibitory effect, 250μg/ml ASODN1 the strongest inhibitory effect (P<0.05), 72h of the role of the effect is stronger than 48h (P<0.05 ); but different concentrations, the difference between ASODN pairwise comparison, the difference was not statistically significant (P>0.05). N-ODN transfected group and the control group and ASODN1, ASODN2 compared with analysis of variance in all experimental groups, the differences were significant (P <0.01 or P<0.05). Multiple comparison among the means was significant(P<0.05).Conclusions1.MK protein and mRNA expression in cervical squamous cell carcinoma of the occurrence and development of about.2. Hela229 cells exist in MK protein and mRNA. Showed that MK can be used as an effective cervical cancer and other cancer gene therapy targets.3. MK ASODN right Hela229 cell protein and mRNA expression of MK significantly inhibited, the results of the study for the application of MK ASODN reach targeted therapy of cervical cancer and other malignancies provide a theoretical basis. |
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