| Objectives:To study the treatment role of recombinant adeno-associated virus (rAAV)mediated rat insulin-like growth factor-1(IGF-1) gene on streptozotocin-induceddiabetic cystopathy, application of the virus vectors for the treatment of diabeticcystopathy lay the foundation of clinical trials in future.Methods:1. Construction of rAAV mediated IGF-1gene vector (rAAV2/1-IGF-1), genomicparticle titer of1.3×1012v.g/ml.2. Diabetic rat model was induced by a single intraperitoneal injection of STZ(65mg/kg body weight) in male Sprague-Dawley rats. Rats were divided randomlyinto two age-matched groups: normal control (n=10) and diabetes groups (n=10),every two weeks for measuring weight and blood glucose level. Rats of two groupswere placed in metabolic cages to measure24h urine output at8weeks after theinduction of diabetes, filling cystometry was taken to obtain urodynamic parametersin order to evaluate the bladder function. We utilized conventional HE stainingmethods to observe the morphological changes of bladder detrusor, and to study thechanges of bladder structure and function and its pathological basis in diabetic rats.3. Rats were divided randomly into five age-matched groups: normal control (CNgroup, n=8), diabetes model (DM group, n=8), simple transduction rAAV2/1-IGF-1model (IGF-1group, n=8), insulin therapy model (INS group, n=8), transductionrAAV2/1-IGF-1&insulin therapy model group (IGF-1+INS group, n=8).8weeks after the induction of diabetes, giving INS group and IGF-1+INS group dailysubcutaneous injection of insulin6IU/rat, IGF-1group and IGF-1+INS groupintramusclar injection of rAAV2/1-IGF-1100μl/rat, respectively. Rats of five groupswere placed in metabolic cages to measure24h urine output after gene therapy6weeks, filling cystometry was taken to evaluate the bladder function. We utilizedconventional HE staining methods to observe the morphological changes of bladderdetrusor. We evaluated the expression profile of IGF-1in rat bladder byimmunohistochemistry (IHS) assay. Study the treatment role of IGF-1gene onstreptozotocin-induced diabetes cystopathy.Results:1. Compared with normal control group, blood glucose level and bladder weightand24h urine volume of diabetic group increased, while body weight decreased (P﹤0.01). The result of cystometry revealed that after8weeks of injection of STZ,bladder capacities of normal and diabetic group were (0.72±0.05) and (2.52±0.24) mlwith significant difference. Post voiding volumes of two groups were (0.05±0.01) and(0.43±0.06) ml with significant difference. Single voiding volumes of two groupswere (0.67±0.05) and (2.09±0.23) ml with significant difference. Voiding efficienciesof two groups were (92.90±0.91) and (82.75±2.54)%with significant difference.Maximum bladder pressures of two groups were (26.92±0.60) and (35.19±1.46)mmHg with significant difference. HE staining found diabetic rats detrusor musclebundles derangement, loose structure, muscle bundles rupture.2. Compared with CN group, four other groups of glucose level and bladderweight and24h urine volume were significantly increased, while body weight wassignificantly decreased (P﹤0.01). Compared with DM group, IGF-1+INS groupblood glucose was significantly decreased, body weight significantly increased (P﹤0.01), bladder weight decreased (P﹤0.05). The result of cystometry revealed thatbladder capacities of group CN, DM, IGF-1, INS, IGF-1+INS were (0.75±0.03),(3.31±0.41),(2.88±0.29),(2.90±0.16) and (1.96±0.57) ml, respectively, withsignificant difference. Single voiding volumes of each group were (0.70±0.03),(2.52±0.29),(2.24±0.18),(2.30±0.14) and (1.70±0.51) ml. Post voiding volumes of each group were (0.05±0.01),(0.79±0.14),(0.64±0.11),(0.59±0.06) and (0.26±0.06)ml. Voiding efficiencies of each group were (92.84±0.67),(76.16±1.87),(77.88±1.82),(79.18±1.34) and (86.59±1.20)%, respectively, with significant difference. Maximumbladder pressures of each group were (27.20±1.00),(35.64±1.74),(34.48±1.73),(34.31±1.06) and (34.72±1.71) mmHg, compared with DM group, no significantdifference was observed among bladder pressures of three treatment groups (P﹥0.05).Diabetic rat bladder pressure curve has many small pressure fluctuation, irregularcurves, and three treatment groups without these performances. Routine HE stainingfound normal control rats detrusor muscle bundles are arranged in rows, compactstructure, the gap is filled with connective tissue, detrusor cell size in homogeneousform. Diabetic rats bladder detrusor muscle bundles derangement, loose structure,muscle bundles rupture, muscle bundles gap widened obviously edama, detrusor cellatrophy, morphological diversity, lymphocyte infiltration, the emergence of a largenumber of vacuolar degeneration, a small amount of acidophilic degeneration,interstitial and collagen components increased. The IGF-1group showed detrusormuscle bundles are arranged in rows, structure more closely, the gap is filled withconnective tissue, muscle cell vacuolation, a small amount of lymphocyte infiltration.The INS group showed detrusor muscle bundles are arranged in rows, structure moreclosely, the gap is filled with connective tissue, a small amount of vacuolardegeneration, muscle cells without atrophy. IGF-1+INS group showed detrusormuscle bundles are arranged in rows, structure more closely, the gap is filled withconnective tissue, muscle cells without atrophy. The results of immunohistochemicalshowed that IGF-1in each group positive staining, and the expression of IGF-1wasfound only in bladder detrusor cell nuclear. Expression of IGF-1protein levels isdown-regulated in diabetic rat bladder. Compared with diabetic rats, three treatmentgroups showed the expression of IGF-1protein enhancement.Conclusions:1. STZ-DM in rats, which has similar pathological and functional changes ashumans, is a reliable and useful model for research on diabetic cystopathy.2. Expression of IGF-1was decreased may be involved in the occurrence and development of diabetic cystopathy.3. rAAV2/1-IGF-1combined with INS therapy can effectively improve bladderfunction of diabetic rats through up-regulating expression levels of IGF-1protein andmay become a reasonable approach for treating diabetic cystopathy. |
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