ABSTRACT
Objective To observe the effects of green fluorescence protein mediated by lentivirus in bladder, and to de?termine the amount of virus obtained good transfection effects. Methods lentivirus carring GFP gene was perfused using transurethral approach into bladder of guinea pigs. Samples of bladder, liver, kidney and lungs were collected for frozen sec?tions after feeding seven days. The distribution of green fluorescence was observed using laser confocal microscopy. Re?sults The titer of lentivirus was 4 × 108. GFP was found under the mucosa when the amount of lentivirus transurethral was 30μL. GFP was distributed widely in muscle layer using 40μL lentivirus. GFP was detected even stronger in muscle layer when the amount was 50μL. GFP was found in muscle layer when 25μL lentivirus was injected intravenously. GFP was not found in other tissues than in bladder via transurethral perfusion. There was higher GFP in liver, lungs and other organs than in bladder via intravenous injection. Conclusion Lentivirus can mediate GFP transfecting bladder of guinea pig successful?ly and escape the distribution of GFP all over the body intravenously, which will bring new research direction and method for clinical treatment of diseases in bladder.
ABSTRACT
Objective To study the beneficial effect and mechanism of Alcea rosea roots in nephrolithiasis model in-duced by 1%ethylene glycol in rats. Methods We randomly divided 60 male Wistar rats into six groups,including control group, model group, Alcea rosea roots lower-dose preventive group, Alcea rosea roots high-dose preventive group, Alcea ro-sea roots lower-dose curative group, Alcea rosea roots high-dose curative group. Control group was free to access food and water;model group was given 1%ethylene glycol drinking water and was fed with normal diet, preventive group was given 1%ethylene glycol drink and Alcea rosea roots in low (250 mg/kg) or high dose (500 mg/kg) each day, curative group re-ceived 1%ethylene glycol drink each day and Alcea rosea roots in low or high dose from day 15 to day 28. At the end of the experiment, various renal functional and injury markers such as urine volume, calcium, phosphate, magnesium, urea, creati-nine, and oxalate were evaluated using urine, serum, and kidney homogenates. The kidneys were removed and prepared for histological evaluation of calcium oxalate deposits. Results In model groups, urine output, urea, creatinine, 24 h urine Ca2+, and oxalate and MDA were increased compared to those in control group(P＜0.05). GSH and SOD were increased in preventative and curative groups compared to those in the model group(P ＜ 0.05). The urea, creatinine, 24 h urine Ca2+, urine oxalate, MDA were reduced in preventive and curative groups compared to those in the model group(P＜0.05). The number and size of calcium oxalate crystal deposits were also less and smaller, and the kidney damage was less severe in pre-ventive and curative groups compared to in the model group. Conclusion The extract of Alcea rosea roots can prevent and treat calcium oxalate urinary stone formation in rats and protect renal function.
ABSTRACT
Objective To investigate the expressions of calcium sensitive receptor (CaSR) and tight junction protein (Claudin)-14 in renal calcium oxalate stone rat model. Methods Thirty male SD rats were randomly divided into control group (n=15) and model group (n=15). The rat model of renal calcium oxalate stone was established by gavaging 1%glycol (2 mL/d) and 2% ammonium chloride. The expression of CaSR protein was detected using immunohistochemical assay. RT-PCR was used to detect the Claudin-14 mRNA expression. The expression levels of CaSR and Claudin-14 protein were de-tected by Western blot assay respectively. The full automatic biochemical analyzer was used to detect rat renal function and changes of blood and urine biochemical indices. Results A large stone crystallization was observed under light microscope in model group. The serum levels of Cr, BUN, 24-h urine calcium and urine volume were significantly higher in model group than those of control group (P<0.01). There were no significant differences in serum calcium level and urinary pH value be-tween two groups. The expression levels of Claudin-14 mRNA and CaSR protein were significantly higher in model group than those of control group (P<0.01). The Claudin-14 protein expression was specifically higher in renal tissues of model group. There was no Claudin-14 protein expression in control group. There was a positive correlation between CaSR and Claudin-14 protein expression in renal tissues of model group. Also, CaSR and Claudin-14 protein expressions were posi-tively correlated with the 24-h urine volume. Conclusion The increased expressions of CaSR and Claudin-14 involved in the formation of kidney stone by increasing the urinary calcium excretion.
ABSTRACT
Objective The calcium oxalate stone is the most common type of the kidney stones.By building the rat renal calcium oxalate stone model, preliminary study the function of CaSR-Claudin14 regulating pathways on renal calcium oxalate stone for-mation model in rats. Methods 30 Male S-D rats were randomly divided into control group (n=15) and model group (n=15). Adult male S-D rats were given ethylene glycol and ammonium chloride to induce urolithiasis.Application of full automatic biochemical analyzer to test rat renal function and the changes of urine biochemical index.Immunohistochemistry was used to detect the expression of CaSR protein;RT-PCR was used to detect the Claudin-14 mRNA expression;Western Blotting was used to detect the expression of CaSR and Claudin-14 protein respectively. Results By observing model group has large stones crystallization under light microsco-py;model group rats 24 h urine calcium are significantly higher than control group([9.66 ±1.10]mmol vs [3.26 ±0.60]mmol, P0.05 ) .Expression of Claudin-14 mRNA in the model group is significantly higher than normal control group([0.150 ± 0.004] vs [0.047 ±0.008], P<0.01); Expression of Claudin-14 protein in the model group is significantly higher than normal control group([1.526 ±0.089] vs 0, P<0.01).Expression of CaSR protein in the model group is significantly higher than normal control group([6.697 ±0.051] vs [5.016 ±0.053], P<0.05). Conclusion CaSR can raise the expression of Claudin-14, increase re-nal tubular urinary calcium excretion to promote renal calcium oxalate stone formation.
ABSTRACT
AIM: To determine the expression of c-kit mRNA and protein in the bladders in guinea pigs with diabetic cystopathy (DCP) and to explore the correlation and mechanisms between c-kit expression and DCP. METHODS: Sixty guinea pigs were divided randomly into control group (n=20) and experimental group (n=40). The guinea pigs in experimental group were injected with streptozotocin (STZ) to induce diabetes mellitus. After fed for 10 weeks, the animals in both groups were tested with urodynamics, and the guinea pigs in experimental group were divided into the subgroups of DCP and the diabetic no-cystopathy (NDCP) group according to the results of urodynamics. mRNA expression of c-kit was detected by reverse transcription polymerase chain reaction (RT-PCR) and protein expression of c-kit was tested and analyzed by laser scanning confocal microscope. RESULTS: Decreased expression of c-kit mRNA was observed in DCP group compared to control and the NDCP group. The ratio of c-kit mRNA and GAPDH was 5.66±0.54 in controls (P<0.05), 5.54±1.28 in NDCP group (P<0.05) and 4.65 ±0.47 in DCP group. c-kit protein expression significantly declined in DCP group. The mean value of fluorescence intensity was 856.52± 53.03 in control group, 844.67± 59.24 in NDCP group and 548.69± 48.51 in DCP (P<0.01).CONCLUSION: The declined expression of c-kit) gene at transcription and translation levels destroys the SCF/c-kit signal pathway, leading to the dysfunction of Cajal-like) cells in DCP guinea pig, so the abnormal expression of c-kit gene is involved in the pathogenesis of DCP.
ABSTRACT
Objective To discuss the types of ICC and characteristics of spontaneous Ca2+ waves of different types of ICC in the bladder of guinea pig. Methods Frozen-sections were made from the bladder of guinea pig and ICC were cultured in vitro. Cells were stained by indirect immunofluorescent method and detected by Laser scanning confocal microscope. The ICC cultured in vitro were divided randomly into 4 group: dimmer ICC,monomer ICC,dimmer ICC treated with 2-APB group and monomer ICC treated with 2-APB group according to the cell morphology and disrupted with 2-APB.The calcium concentration of ICC cultured in vitro were marked with Fluo-4 AM and disrupted by 2-aminoethoxydipheylbrate (2-APB, 100 μmol/L) in dimmer ICC treated with 2-APB group and monomer ICC treated with 2-APB group. The calcium oscillation function of ICC was observed under Laser scanning confocal microscope. Results For the monomer ICC and dimmer ICC in frozen sections and cultured in vitro,there were increased frequency (P＜0.01) and amplitude (P＜0.05) of spontaneous Ca2+ waves in dimmer ICC compared with the monomer monomer ICC. But after the cells disrupted by 2-APB after 15 min,There were decreased frequency (P＜0.01) and amplitude (P＜0.01) of spontaneous Ca2+ waves in the dimmer ICC treated with 2-ABP group compared with the dimmer ICC. The changes(P＞0.05) of spontaneous Ca2+ waves was not statistical significance in monomer ICC treated with 2-ABP group compared with monomer ICC group. Conclusions The bladder of guinea pig may exist 2 different types of ICC, dimmer ICC and monomer ICC. The excitability of spontaneous Ca2+ waves of dimmer ICC could be higher than in monomer ICC. The special structure of dimmer ICC may contribute to the formation of high spontaneous Ca2+ waves.
ABSTRACT
Objective To determine vascular endothelial growth factor(VEGF)-460 gene polymorphism in Uyghurs and its relationship to urolithiasis in south Xinjiang. Methods Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP),gene sequencing and genetic analysis methods were used in 200 urolithiasis patients of Uyghurs, and 200 healthy Uyghurs. Results The distribution of genotype and allele had no significant difference between urolithiasis patients and normal controls (P＞0. 05). The frequencies for the CC,TT and CT genotypes in patients with urolithiasis and normal controls were 1.5 %, 29.0 %, 69.5 % and 0. 5 %, 27.5 %, 72.0 %, respectively. The frequencies for C and T allele were 36.2%,63.7% and 36.9% ,63.1%, respectively. Conclusions The results of VEGF-460 gene polymorphisms indicate no significant relationship between patients with turolithiasis and normal controls in Uyghurs in south Xinjiang,which may not be urolithiasis susceptibility genetic locus.
ABSTRACT
Purpose To clarify the morphologic changes and significance of interstitial cells of Cajal-like cells in the bladder of diabetic guinea pig.Methods The experimental guinea pigs were induced by a single intraperitoneal injection of streptozotocin(STZ).The diabetic guinea pig model, who were made succesful for 4 weeks, were detected through the urodynamic evaluation.The distribution of ICCs-like cells in the detrusor, which were taken from these guinea pigs,were quantitatively analyzed after frozen section and indirect immunofluorescence methods and observed by electron microscopy.Results In the diabetic group, the maximum detrusor pressure was signifently lower than controls (P＜0.01), maximum bladder capacity, compliance, residual volume and leak point pressures was significently higer than controls (P＜0.05 or P＜0.01);In detrusor, compared with control, the ICCs-like cells of diabetes group showed significently decreased in quantity. In diabetic bladder, the quantity of gap junctions between ICCs-like cells and cells around,such as smoth muscle cells,terminal nerves and other ICCs-like cells,decreased significently.And more cavities and less organoids occurred in cytoplasm of diabetic ICCs-like cells. Mitochondria became swollen or even disapeared, accompanied with swollen endocytoplasmic reticulum.Conclusion The decrease and injures of ICCs-like cells in bladder may be one of the mechanisms resulting in urodynamic changes of diabetic cystopathy.
ABSTRACT
Objective To investigate the expression level of cyclooxygenase-2 (COX-2) and its effects on cell proliferation and apoptosis in bladder cancer. Methods The immunohistochemical method was used to determine the expression of COX-2 and Ki67 in 54 cases of bladder cancer, 29 cases of adjacent tumor tissue and 10 cases of normal bladder specimen respectively, and the apoptotic levels of the obove three tissues were assessed by counting the number of the cells labeled by TUNEL method. Results The positive rate of COX-2, the mean labelling index of Ki67 and the cellular apoptosis index in the cancer tissues were significantly higher than those in adjacent tumor tissues and normal bladder specimens(P
ABSTRACT
Objective To study the correlation of NF-?B expression with the grade, the stage, the recurrence, the metastasis and PKC expression of bladder transitional cell carcinoma. Methods The expression of NF-?B and PKC was examined with SP immunohistochemical method in 54 cases of paraffin-embedded bladder transitional cell carcinomas tissues and 13 cases of normal bladder tissues. Results The positive expression rate of NF-?B P65 and PKC in bladder transitional cell carcinomas was 88 9% and 81 5% respectively. The expression rate of NF-?B decreased along with pathological grade and clinical stage increased, while its expression increased along with the rate of recurrence and metastasis elevated. There was a positive relationship between NF-?B and PKC expressions. Conclussion NF-?B may play a role at early stage of bladder transitional cell carcinoma. It probably serves as an index to evaluate the malignant extent and progression of bladder transitional cell carcinoma. PKC probably takes part in the activation process of NF-?B.
ABSTRACT
Objective To identify the putative CpG-N ODNs in adenovisus 2 DNA (Adv2 DNA) and Adv5 DNA by comparing the sequence difference among Adv2, 5, 12 DNA and E.coli (EC) DNA. Methods Sequences of Adv2, 5, 12 DNA and EC DNA were obtained from the Entrez Nucleotides database at NCBI. The specific CpG motifs of Adv2 DNA and Adv5 DNA were identified after above sequences were analyzed and compared by softwares such as DNATools, BioEdit, and so on. All the 12-ODNs with specific CpG motif core were searched from Adv2 DNA and Adv5 DNA. Results 19 specific CpG motifs were ascertained and 504 12-ODNs were detected in Adv2 DNA and Adv5 DNA. Conclusion 504 12-ODNs were putative CpG-N ODNs in Adv2 DNA and Adv5 DNA.
ABSTRACT
Objective To obtain a potent bacterial DNA antagonizing CpG oligonucleotides (CpG-N ODN) from the structures of Adv2, 5 DNA. Methods Ten putative CpG-N ODNs were synthesized and investigated. Their abilities to inhibit the TNF-? release from hPBMC were observed. Based on the above results, the putative CpG-N ODN was redesigned according to the relationship between the structure and free energy using RNA structure software (version 3.71). Eleven putative CpG-N ODNs were synthesized and screened. Results Out of the ten initial CpG ODNs, ODN101 was the only CpG-N ODN with weak activity to inhibit TNF-? release from hPBMC induced by CpG-N ODN. After redesign, five CpG-N ODNs with strong activity were confirmed. Conclusion Six CpG-N ODNs were obtained with activity to inhibit TNF-? release from hPBMC induced by CpG-N ODN.