Expression Of IGF-1 MRNA In Rat Bladder Of Diabetic Cystopathy

Objective: To observe the IGF-1 (Insulin-like growth factor-1) mRNA expression in diabetic rat bladder with Real-time PCR technique and to further explain the expression of IGF-1 changes in the role of Diabetic cystopathy.Diabetic cystopathy(DCP) is a kind of diabetes complications of urinary system, diabetic peripheral neuropathy is the main reason. Onset of bladder dysfunction has nothing to do with age, sex. The disease does not have early obvious symptom, so early diagnosis is difficult. Its clinical characteristics is urine output and micturition frequency change, dysuria, urinary incontinence, bladder capacity increases, appear residual urine and urinary tract infections. Diabetes cystipathy does not have the special effect method of treatment at present, treatment is still mainly based on treatment of diabetes, and supplemented by other therapeutic measures. The treatment for early diabetic cystopathy can delay the progression, but this method in patients with advanced bladder symptoms can not be effectively reversed. Lead to poor quality of life of patients with advanced. Therefore, diabetes cystipathy's early diagnosis, the treatment and the cause of disease research has become the key point which the researchers pay attention. In recent years, along with molecular biology, cytobiology, cytophysiology and nerve biology, nerve physiology development progress, especially in the nerve injury repair and prevention and treatment of neurodegenerative diseases in-depth research. On the development of some neural differentiation and regeneration are closely related biologically active substances, such as insulin growth factor (IGF), nerve growth factor (NGF), carried out extensive and in-depth study, both in theory and applications has made considerable progress. Recent studies showed that insulin-like growth -Ⅰ(IGF-1) is a kind of variety of biological functions of cytokines, can promote cell differentiation, proliferation, inhibition of apoptosis. Especially, IGF-1 to the proliferation and differentiation of nerve cells,Osteoblasts,etc, play a key regulatory role,contribute to neurological functional recovery after nerve cells damage. Has now been confirmed on the nervous system plays an important protective effect of nutrition. In the diabetic cystopathy, the current home and abroad to focus on the NGF and diabetic cystopathy relations. At present about IGF - 1 and diabetic cystopathy is less, the research of the relationship is in its infancy stage. Real-time PCR in the detection of IGF-1 factor has inherent advantages. Compared with other experimental method, it's not only simple operation, but also more objective and accurate. This method of RNA quality requirements is still very low, can detect very low concentrations of mRNA.This study provide experimental basis for research and treatment of diabetic cystopathy, also provide a new and effective way for researching diabetic cystopathy.Methods:1 Experimental animals and groups: 30 Sprague-Dawley (SD) male rats from the Experimental Animal Center of Hebei Medical University were used, weighing 200 ~ 250g. Animals were andomly divided into 3 groups, normal control group, diabetic group, treatment group, n = 10. SD rats were fasted for 12h. The diabetic group and the treatment group rats were injected intraperitoneally 60mg/kg STZ. The control group rats were injected intraperitoneally isopyknic citrate buffer. After injection STZ 72 hours, measuring fasting blood glucose. BG> 16.7mmol / L as a successful model. According to the fasting blood glucose, they were randomly divided into diabetes group and treatment group. SD rats of treatment group were given subcutaneous injection of protamine zinc insulin at the day after the model successfully, so that BG remained at 3.9 ~ 10mmol / L. Normal control group and diabetes group also given the same volume of saline subcutaneously. Three groups of the experiment is in 8 weeks after the modeling. 2 Real-time PCR(Real-time quantitatie polymerase chain reaction): RNA extraction Reagent is TRNzol Reagent from TIANGEN BIOTECH (BEIJING)Co., Ltd,according to instruction booklet operation. Quantscript RT Kit is come from TIANGEN BIOTECH(BEIJING)Co., Ltd, according to instruction booklet operation. Internal reference gene isβ-actin. Primer is come from Sangon Biotech(shanghai)Co.,Ltd. Real-time PCR reaction system is used in 20ul, including: 2.5×RealMasterMix/20×SYBR solution 9ul, cDNA 2ul, Primer 0.5ul, Ultra-pure water 8ul. Reaction in ABI7500 fluorescence quantitative polymerase chain reaction (PCR) instrument. Reaction conditions: 95℃initial template denaturation for 2 minutes, 1 cycle. 95℃PCR template denaturation for 20 seconds, 60℃annealing for 30 seconds, 68℃extensions for 50 seconds, 40 cycles, the detection of fluorescence at 68℃. Analysis software through the fluorescence intensity get sample IGF-1,β-actin Ct values.3 Statistical analysis: According to IGF-1,β-actin in the Ct values, using 2-△△Ct method, The△Ct = Ct IGF-1 (target gene) - Ctβ-actin (internal reference gene) express the relative gene copy number.△Ct the value lower prompt gene expression quantity is higher. Gene expression quantity of the IGF-1andβ-actin is expressed by the mean value±standard deviation method. Statistical ANOVA to P <0.05 with a statistically significant, with SPSS18.0 packages completed.Results:Rat bladder tissue expression of IGF-1 mRNA: The bladder tissue total RNA of diabetic group, treatment group and normal control group was Real time PCR (real-time quantitative PCR),△C t values is in Table 1. IGF-1 mRNA expression in the diabetic group was 6.141±0.916, the expression of the normal control group was 4.018±0.491, expression in the treatment group was 4.922±0.788. The data were statistically analyzed (Table 2). IGF-1 mRNA expression in the diabetic group was significantly lower (p <0.05), Up-regulated by insulin treatment of IGF-1 mRNA expression (p <0.05, Fig.1). Conclusion:This experiment through Real-time PCR research IGF-1 mRNA in diabetic rat bladder 's expression change , the following conclusions:1 IGF-1mRNA expression in diabetic rat bladder was significantly reduced , participated in diabetes cystipathy's pathological change.2 The insulin may reverse IGF-1 mRNA in diabetic rat bladder's expression in treatment group, suggesting that No relationship between IGF-1mRNA significantly reduced and the toxicity of streptozotocin.