Objective To establish a method of isolating,culturing,proliferating urinary tract(nephropelvics,ureter and bladder)smooth muscle cells (SMC). Methods Urinary tract SMC were isolated from sample tissues (1 from human nephropelvics,1 from human ureter,3 from human bladders,5 from porcine bladders and 1 from porcine ureter) by collagenase digestion and were cultured in DMEM supplemented with fetal bovine serum.Morphology and expansion of the cells were observed.SMC specific protein (?-actin) was identified by immunohistochemical methods. Results The cells grew well and presented a typical morphological feature of SMC.They could be passaged 6 times without a remarkable decrease in rate of cell proliferation.Immunohistochemical staining showed that ?-actin was positive. Conclusions This urinary tract SMC cultural method was simple,reliable and fruitful.

Objectives To determine the causes of cubital tunnel syndrome by performing operation,and to assess the clinical value of neurophysiologic diagnosis in cubital tunnel syndrome Methods Twenty one patients (22 limbs from 16 men and 5 women, aged from 22 to 63 years, with mean age of 49 years)suspected of ulnar nerve involved in clinical symptoms and the signs of ulnar distribution were recorded with motor conduction velocity at different sites along with the ulnar nerve and sensory conduction velocity in the hand,and underwent anterior transposition of the ulnar nerve Results Electromyographic abnormality was seen in 21/22 (range MCV 15.9～47.5 m/s,mean 32.7 m/s) of patients with motor conduction velocity in across elbow segment of the ulnar nerve. And slowing was seen in 13/22 (MCV 15.7～59.6 m/s, mean 40.4 m/s )patients with MCV in the forearms. The absent or abnormal evoked sensory nerve action potential was seen in 14/22 patients in the little finger. The factors of ulnar compressed in this study by operation were:15 ulnar nerves compressed by arcuate ligaments, or muscle tendons, or bone hyperplasia; two were involved in fibrous by adhesion;three compressed by venous plexus or concurrent thick vein;two compressed by cysts Conclusions The factors of cubital tunnel syndrome include either the common factors reported or the rare factors, such as the venous plexus, thick vein and cysts. The tests of motor conduction velocity at different sites along the ulnar nerve should be helpful in diagnosis of cubital tunnel syndrome, especially the slowing velocity of MCV across the elbow segment.

ObjectiveTo investigate the clinical feature peripheral neuropathy and vasculopathy after acrylamide toxication. Methods2 young male patients with peripheral neuropathy who had exposed to acrylamide for job more than one year were reported.ResultsNeuroelectrophysiological examination showed marked abnormalities in both peripheral and central nerve conduction in both patients. Sural nerve biopsies revealed axonal degeneration, Wallerian degeneration and giant axon with accumulated neurofilaments. Additonally, vasculopathies including prominant thickness of arterial intesma and basal membrane of capillary as well as apoptosis of vascular pericyte, were evident. ConclusionAxonal degeneration and vascular involvement has been found in acrylamide toxication. Vascular impairment maybe plays an important role in the pathogenesis of neuropathy.

Objective To investigate the significance of quantitative thermal testing (QTT) in the diagnosis of diabetic peripheral neuropathy. Methods One hundred and sixty-nine diabetic patients with neurological deficit (DM group) and 53 age-matched healthy controls underwent the determination of cold threshold (CT), warm threshold (WT), clod pain threshold (CPT), warm pain threshold (WPT) in both dorsum of hand and dorsum of foot. DM group were divided into subgroups with a course of disease > 5 years or with a course of disease ≤ 5 years, or divided into subgroups with normal or abnormal nerve conduction study (NCS). Results CT and WT of DM group with a course of disease ≤ 5 years ((29.6 ± 1.4), (26. 5±4. 3) ℃ ; (35.9±3.0), (41.3±4. 0) ℃) were higher than the health controls' ((30. 2±1.2), (29.1±1.5) ℃; (35.0±1.9), (36.5±1.5) ℃, respectively; t=3.27, 6.63, 2.80, 8.61, all P< 0. 05). The CT and WT of DM group with a course of disease > 5 years' ((28. 2±4. 0), (23. 1 ±7.9) ℃ ; (37.0±4. 7), (42. 6±4. 2) ℃, respectively) were higher than the DM group with course of disease≤ 5 years(t =4. 09, 4.63, 2.55, 2. 68 ,all P <0. 05). CT and WT of the normal NCS group((29. 5 ± 1.8), (27.0±4. 6) ℃ ; (35.0±1.9), (40. 9±3. 8)℃, respectively) were higher than the healthy controls' , and the difference was significant(t =3.22, 4. 17, 3.51,9. 95,all P<0.01). The frequency of abnormal QTT in DM group was higher than that of NCS in DM group. The QTT and NCS of DM group with a course of disease >5 years were higher than these in DM group with a course of disease ≤5 years; the frequency of abnormal WT in DM group(86. 4% ,146/169)was higher than that of CT in DM group(68. 1% ,115/169,x2=15.49, P<0.01), the frequency of abnormal QTT in the dorsum of foot in DM group was higher than that in the dorsum of hand in the DM group. PT of diabetic patients were higher than that in the healthy controls. Condusions QTT is more sensitive than NCS in the diagnosis diabetic peripheral neuropathy, which is neeossary to assist NCS when diabetic peripheral neuropathy is suspected, WT in dorsum of foot is a sensitive parameter in the diagnosis of diabetic peripheral neuropathy.

ObjectiveTo investigate the clinical and pathological features of Hirschprung disease (HD), intestinal neuro-nal dysplasia (IND) and hypoganglionosis (IH) in children.MethodsThe clinical data and pathologic slices from 238 children with intestinal dysganglionosis were retrospectively analyzed. The age, sex, involved intestinal length of children and prognosis were compared.ResultsIn 238 patients, 138 (58.0%) were diagnosed by rectal mucosal biopsies. There were 122 HD patients whose median age at diagnosis was 9 months and the ratio of male to female was 4.3:1, without involvement of whole colon. There were 45 IND patients whose median age at diagnosis was 14 months and the ratio of male to female was 1.05:1, and the whole colon of 33.3% patients was involved. There were two male IH patients whose ages at diagnosis were 12 years and 18 years respectively, and their whole colon was involved. There were 59 patients with HD complicated by IND whose median age at diagnosis was 13 months and the ratio of male to female was 5.56:1 and the whole colon of 16.9% patients was involved. There were 10 male patients with HD complicated by IH whose median age at diagnosis was 11.5 months and the whole colon of 80.0% patients was involved. The ages at diagnosis, the sex ratio, the rates of whole colon involved, and the cure rates among 5 groups were signiifcantly different (allP<0.01).ConclusionsThe rectal mucosal biopsy was the main method in diagnosis of intestinal dysganglionsis in children. Patients with HD had higher incidence and mild condition and favorable prognosis. Patients with IH or patients with HD complicated by IH had lower incidence rates and severe condition and poor prognosis, followed by patients with IND or patients with HD complicated by IND.

OBJECTIVETo pursue insulin and islet-transplantation replacement therapy for type 1 diabetes based on engineered human non-beta cells which secrete mature insulin.

METHODSHuman proinsulin cDNA was cloned from its genomic gene and mutated by overlap extension PCR, introducing furin consensus cleavage sequences (Arg-Xaa-Lys/Arg-Arg). An expression vector encoding a genetically modified human proinsulin cDNA was generated and transduced to Hela, 293, and L02 cells by lipofectin-mediated DNA transfection. Following G418 screening, the surviving L02 cells were selected and enriched. Insulin levels in the supernatant and cells were evaluated using radioimmunoassay and immunofluorescence staining.

RESULTSThree sites in the insulin gene were mutated simultaneously. Insulin gene modified cells were able to express insulin at different levels: 8.45 - 188.00 microIU/24 h/2.0 x 10(6) Hela cells and 159.88 - 242.14 microIU/24 h/2.0 x 10(6) 293 cells for transient expression, and 2.56 - 61.95 microIU/24 h/2.0 x 10(6) from several L02 clones screened with G418. No insulin was released by control cells. Furthermore, immunofluorescence staining confirmed that proinsulin was stored as vacuoles in the cytoplasm of L02 cells.

CONCLUSIONA correctly mutated human proinsulin cDNA was obtained successfully, transfected and expressed efficiently in non-beta cells, lending support to the study of somatic gene therapy in diabetes mellitus.

Cell Line ; Chromatography, High Pressure Liquid ; DNA, Complementary ; genetics ; Fluorescent Antibody Technique ; Genetic Vectors ; genetics ; HeLa Cells ; Humans ; Insulin ; genetics ; metabolism ; Proinsulin ; genetics ; Radioimmunoassay ; Transfection

ObjectiveTo explore the effect of the serum containing Huanglian Wendantang on pyroptosis in vitro model of insulin resistance and its mechanism. MethodSD rats were randomly divided into two groups， namely Huanglian Wendantang-containing serum group and blank serum group， and given 7.8 g·kg^-1·d^-1 Huanglian Wendantang and equal volume of normal saline by intragastric administration according to body surface area. Blank serum and medicated serum with different concentration were extracted and prepared. HepG2 cells were treated with sodium palmitate to construct the model of insulin resistance （IR）， and they were randomly divided into control group， model group， metformin hydrochloride group， blank serum group， and Huanglian Wendantang-containing serum high-， medium-， and low-dose groups. After 24 h of cultivation， the cells of each group were treated with insulin for 15 min at concentration of 1×10^-7 mol·L^-1， and the cell supernatant was collected. The glucose oxidase （GOD-POD） kit was used to determine the glucose content of each group， and calculate the glucose consumption and inhibition rate. The methyl thiazolyl tetrazolium （MTT） assay was used to detect the cell proliferation， thus screening out the optimal dose of serum containing Huanglian Wendantang. HepG2 cells were randomly divided into control group， model group， and Huanglian Wendantang-containing serum group. The levels of interleukin-1β （IL-1β） and interleukin-18 （IL-18） in each group were determined by enzyme-linked immunosorbent assay （ELISA）， and the mRNA and protein expression levels of NOD like receptor protein 3 （NLRP3） in each group were determined by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot. In terms of the mechanism， HepG2 cells were randomly divided into control group， empty vector group， NLRP3 overexpression group， empty vector + IR group， empty vector + IR + Huanglian Wendantang-containing serum group， NLRP3 overexpression + IR group， and NLRP3 overexpression + IR + Huanglian Wendantang-contain serum group. GOD-POD method was used to measure the glucose content of each group cells， and calculate the glucose consumption. ELISA was used to determine the release of IL-1β and IL-18 in each group. Real-time PCR and Western blot assay were used to determine the mRNA and protein expressions of cysteinyl aspartate specific proteinase-1 （Caspase-1）， gasdermin D （GSDMD）， and NLRP3. Immunofluorescence assay was used to detect NLRP3， GSDMD， and Caspase -1 expressions. ResultAs compared with the control group， the glucose consumption in the model group was significantly decreased （P<0.01）. As compared with the model group， the increase of the glucose consumption of IR-HepG2 cells was the most significant in the Huanglian Wendantang-containing serum high-dose group （P< 0.01）. As compared with the control group， the IL-1β and IL-18 release levels and the mRNA and protein expressions of NLRP3 in IR-HepG2 cells were significantly increased （P<0.05， P<0.01）. Huanglian Wendantang effectively reduced IR-HepG2 cell supernatant IL-1β， IL-18， and NLRP3 mRNA and protein expressions as compared with the model group （P<0.05， P<0.01）. Overexpression of NLRP3 significantly reduced the cell glucose consumption as compared with the control group and the empty vector group （P<0.01）， and significantly up-regulated the IL-1β and IL-18 levels and the mRNA and protein levels of NLRP3， Caspase-1， and GSDMD as compared with the empty vector + IR group （P<0.05， P<0.01）. Huanglian Wendantang-containing serum effectively reversed the above indicators as compared with the NLRP3 + IR group （P<0.05， P<0.01）. ConclusionHigh fat-induced insulin sensitivity of IR-HepG2 cells is closely related to inflammation and NLRP3 expression. Huanglian Wendantang-containing serum improves IR-HepG2 cell pyroptosis through the targeted inhibition of NLRP3/Caspase-1 signaling pathway， which provides new therapeutic targets for the prevention and treatment of IR and type 2 diabetes mellitus （T2DM）.