| Bladder carcinoma has a higher incidence in the world and is the fifth malignant tumor. Besides, bladder carcinoma has the highest incidence and death rate in urinary surgery and its incidence is on the rise year by year, posing serious threat to people’s health. More than 95 percent of bladder carcinoma come from urothelial cells. According to the growth pattern, bladder carcinoma are divided into superficial and invasive growth. In all cases of bladder carcinoma, the majority are superficial tumors in the growth chamber. At present, transurethral resection of bladder carcinoma is the most widely used treatment method. Even aided by intravesical chemotherapy,the recurrence rate is still high. 14% to 45% of the cases develop into invasive bladder carcinoma in the end. For invasive bladder carcinoma, the most widely used treatment is curative resection. In this case, the recurrence rate is about 50 to 70 percent and about half of the patients died after 4 to 5 years. The early diagnosis of bladder carcinoma is significant in improving the therapeutic effect and reducing the death rate.Objective: Normal bladder epithelium tissue and bladder carcinoma tissue of different clinic pathological factors are studied in this paper. The experiment uses immunohistochemistry SP method and Western Blotting method to determine the expression of KLF4 in normal bladder tissue and bladder carcinoma tissue of different clinic pathological factors. The results are statistically analyzed. The purpose is to reveal the relationship between the expression of KLF4 protein and the incidence,development and clinical pathology of bladder cancer:Method: tissue origin: 51 cases of bladder carcinoma tissues and 30 cases of normal bladder epithelium tissue removed from patients in urinary surgery of No. 1 Hospital of Shijiazhuang and PLA 260 Hospital from November 2013- January 2015.All the cases were pathological confirmed the diagnosis of urothelium carcinoma.According to the criterion of the WHO pathology of new classify of tumours in 2004 years,there are 21 high grade cases and 30 low grade cases.There are 33 male patents and 18 female patients, aging from 24 to 79 and the average age is 55.0 years old. All patients did not receive any radiotherapy, chemotherapy or immunotherapy before the resection. The specimens used in immunohistochemical SP method were fixed in 10% formalin and embedded in paraffin wax, and then cut into 4μm thick serial sections. The specimens used in western blot blotting method after surgery was immediately placed into sterilized Eppendof tubes in sterile condition, which was placed in liquid nitrogen and transported to-70 ℃fridge for cryopreservation and standby application.1 Immunohistochemical SP method(two-step): Immunohistochemical SP method is used to test the expressions of the zinc finger transcription factor KLF4 protein in 51 cases of bladder carcinoma tissues and 30 cases of normal bladder epithelium tissue. The expression level of KLF4 in normal bladder epithelium and different clinic pathological factors is calculated. The calculation results are recorded in forms. The experiment results are generated by SPSS 19.0 statistical software. The expressions of KLF4 in carcinoma tissue and normal bladder epithelium are compared to find the expression differences of KLF4 in different clinic pathological parameters. The statistic results are tested by X2 and the indicator for statistically significant difference is P <0.05.2 Western Blotting detection: first the SDS-PAGE gels are prepared. The specimens of normal bladder mucosa and bladder cancer tissue are prepared. Liquid supernatant is extracted and used as specimen, which goes through protein content measurement. The KLF4 protein sample was added in proportion to SDS loading buffer and boiled in 100 ℃until albuminous degeneration. Then it put into electrophoresis of 60 V SDS-PAGE for 3 hours in room temperature. The proteins are transferred onto PVDF membranes. The PVDF membrane was placed in 5% nonfat dry milk for primary and secondary antibody incubation. Finally, chemiluminescence method is used to detect the protein. Western blotting imaging analysis software is used in the quantitative analysis of the results.Result: Immuno-histochemistry results indicated that KLF4 has expressions in both bladder urothelial carcinoma tissues and normal bladder epithelium tissues, and the expressions are diffusive and polarity-loss ones, which take forms of brownish yellow granules, located in the cytoplasm, dispersed, and some cells show nuclear staining. KLF4 has higher expression in normal bladder tissues than in carcinoma tissues. The KLF4 expression intensity in non-invasive bladder carcinoma tissues was significantly higher than that in invasive bladder carcinoma. The KLF4 expression in well-differentiated carcinoma is significantly higher than that in poorly differentiated bladder cancer tissues. There is no significant difference between KLF4 expression in bladder urothelial carcinoma tissues and the age, gender, clinical stage, tumor size, lymph node metastasis and distant metastasis of the patient.Western-blotting analysis indicate that KLF4 has high expression in all the normal bladder epithelium tissues.KLF4 was expressed in 24 cases of carcinoma tissue as 6 of which have no KLF4 expression. The expression of KLF4 was examined by the ratio of electrophoresis strip between KLF4 and β-actin, t-test was conducted on the determination results,shows that KLF4 has higher expression in normal bladder tissues than in carcinoma tissues.Conclusion:1 The expression of KLF4 protein in bladder carcinoma tissues is significantly lower than that in normal bladder epithelium;2 The expression of KLF4 protein in low grade bladder carcinoma tissues is significantly higher than that in high grade bladder carcinoma;3 The expression of KLF4 protein in invasive urothelium carcinoma is lower than that in non- invasive urothelium carcinoma.4 The experiment confirms that KLF4 is closely related to the occurrence and development of bladder urothelium carcinoma. |
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