Evaluation of antisperm antibody in infertile women with polycystic ovary syndrome

AIM: To investigate the level of circulatory antisperm antibody (ASA) in infertile women with polycystic ovary syndrome (PCOS). METHODS: A total of 127 infertile women with PCOS (26~35 years old) were divided into 2 groups according to the body mass index (BMI):obese group (BMI ≥ 25 kg/m², n=42) and non-obese group (18 kg/m² < BMI < 25 kg/m², n=85). The infertile women aged 24~36 years of normal weight without PCOS (n=90) were chosen as controls (control group; 18 kg/m² < BMI < 25 kg/m²). The serum levels of ASA-IgG and ASA-IgA were screened by the indirect immunobead test according to the WHO laboratory manual. The 50% or more of the motile sperm attached to one or more immunobeads were regarded as clinical positivity according to the WHO criteria. The 20%~49% motile sperm that had adherent particles were deemed to be weakly positive. Less than 20% was negative. RESULTS: No case in obese group and non-obese group showed the positive level of ASA-IgG. Two cases in control group were detected to be ASA-IgG positive (2.2%). One case in obese group (2.4%), 2 cases in non-obese group (2.2%) and 2 cases (2.2%) in control group were detected to be ASA-IgG weakly positive, and no difference was found in the weakly-positive incidence among these 3 groups. ASA-IgA was not detected in all the cases. CONCLUSION: The circulatory ASA is not associated with the pathogenesis of infertile women with PCOS. The detection of ASA still needs to be routinely performed for infertile women.     

Changes and clinical significance of serum protein factors in patients with polycystic ovary syndrome

AIM:To study the changes and clinical significance of serum protein factors in the patients with polycystic ovary syndrome (PCOS). METHODS:The relative expression levels of 174 ser protein factors in 5 PCOS patients and 5 control women were assayed simultaneously by Antibody Protein Array 2000,in which 2 factors were chosen to test and reconfirm the results by ELISA in 30 cases of PCOS patients and 30 cases of controls. RESULTS:Among 174 serum protein factors, the factors that elevated in PCOS patients were angiogenin, EGF, IGFBP-1, IGFBP-4, IL-7, MIP-1-delta, PARC, PDGF-BB, Acrp30, EGFR, ICAM-1, sTNF-RI, TIMP-2, TRAIL-R3, E-selectin, IL-1RII, IL-5Ra, LAP, L-selectin, M-CSFR, PDGF-AA, prolactin and VEGFR2, while the factors that decreased in PCOS patients were GRO, IL-8 and Siglec-5. Nine factors were increased 3 times more than the controls(IL-1R-II, prolactin, IL-7, IGFBP-1, IL-5Ra, IGFBP-4, VEGFR2, ICAM-1 and MIP-1-delta). PDGF-BB and Acrp30 were tested by ELISA in 60 samples and there were significant differences between the 2 groups. CONCLUSION: The changes of the protein factors in serum might play an important role in the pathogenesis of PCOS.     

Effects of astragaloside on the levels of sex hormone and oxidative stress injury in rats with polycystic ovary syndrome

AIM To investigate the effects of astragaloside on the levels of sex hormone and oxidative stress in rats with polycystic ovary syndrome （PCOS）. METHODS Female SD rats （n=60） were randomly divided into normal control group, model group, Diane-35 （0.339 2 mg/kg） group, low dose astragaloside （12.5 mg/kg） group and high dose astragaloside （50 mg/kg） group, with 12 rats in each group. The PCOS model was induced by letrozole （1 mg/kg）, which was administered by gavage once a day for 3 weeks. After administration, the estrus cycle of the rats was observed by vaginal smear, and the ovarian index was calculated. HE staining was used to observe the histopathological changes of the ovaries. Serum levels of the sex hormones testosterone （T）, estradiol （E₂）, luteinizing hormone （LH） and follicle-stimulating hormone （FSH） were measured by ELISA. The levels of superoxide dismutase （SOD）, malondialdehyde （MDA） and glutathione peroxidase （GSH-Px） in serum and ovarian tissue were detected by colorimetry, and the protein levels of steroidogenetic acute regulatory protein （StAR） and apoptosis-related proteins cleaved caspase-3, Bax and Bcl-2 in ovarian tissue were detected by Western blot. RESULT Compared with control group, the oestrous cycle of the rats in model group was disorder, and the ovarian index was increased, ovary was polycystic. The serum levels of T, LH and MDAwere significantly increased （P<0.05）, while the contents of E2, FSH and the activities of GSH-Px and SOD were significantly decreased （P<0.05）. The levels of MDA, StAR, cleaved caspase-3 and Bax proteins in ovarian tissue were significantly up-regulated （P<0.05）. GSH-Px and SOD activities and Bcl-2 protein levels were significantly down-regulated （P<0.05）. CONCLUSION Astragalosideeffectively balances the levels of sex hormone in PCOS rats and relieves the oxidative stress injury, the mechanism may be related to the inhibition of StAR expression.     

Premature response to luteinizing hormone of granulosa cells from women with polycystic ovary syndrome

AIM:To demonstrate the relationship between hormones in follicular fluid and the expression of LH receptor in granulosa cells (GC) in anovulatory women with polycystic ovary syndrome (PCOS). METHODS:Follicles were obtained from 12 women with PCOS and 15 women with normal menstrual period through surgery at time between day 7 and day 10 of menstrual cycle. The accumulations of estrogen (E2), progesterone (P), luteinizing hormone (LH), follicle stimulating hormone (FSH) and insulin in follicular fluid were determined by a automatism chemiluminescent microparticle immunoassay (CMIA) for the quantitative determination. The accumulation of androstenediol (A) was determined by ELISA. The amounts of the mRNA expressions of LH receptors from GC and theca cells (TC) respectively were measured by RT-PCR using β-actin as intra-control simultaneously. RESULTS:The levels of LH ［(3.8±2.1 vs 1.7±0.8)IU/L, P<0.01］, A ［(600.0±373.4 vs 212.4±205.4)μg/L, P<0.05］ and expressions of LH receptor mRNA of GC (0.29±0.16 vs 0.12±0.13, P<0.01) and TC (0.46±0.14 vs 0.34±0.09，P<0.05) in the women of PCOS group were statically higher than those in control group. The expression of LH receptor mRNA was not detected by RT-PCR in control group when the diameter of an follicle was less than 7 mm, while it was detected in women with PCOS when it remained as small as 4 mm. Expression of LH receptor mRNA in granulose cells was positive related to the concentration of LH (r=0.67, P<0.01) and insulin (r=0.51, P<0.05) in follicular fluid, and that in theca cells (r=0.60, P<0.01). CONCLUSION:The high level of LH in follicular fluid occurs and GC responds to LH prematurely and more intensively in anovulatory women with PCOS. Larger amount of A and P was produced as a result. All of above may contribute to the mechanism of anovulatory.     

High-fat diet affects plasma membrane GLUT4 content in skeletal muscle from Sprague-Dawley rats

AIM: To explore the change of the amount of GLUT4 protein at the plasma membrane of the rat skeletal muscle after high-fat feeding. METHODS: The animals were divided into three groups (ten for each): group I: control; group II: high-fat feeding; group III: high-fat feeding + dietary treatment. The rat model of insulin resistance (IR) was made by feeding high-fat diet for eight weeks. And then insulin-resistant rats were fed with chow diet for 4 weeks. Fasting plasma glucose and fasting serum insulin levels were measured before and after dietary treatment, respectively. Insulin treatment was achieved by intraperitoneal injection of insulin (10 unit insulin per kg body weight) 15 minutes before killing the animals. The right hindlimb skeletal muscle was rapidly dissected. Then the expression of GLUT4 protein at the plasma membrane in all the animals was assessed with Western bloting. RESULTS: The GLUT4 content at the plasma membrane in high-fat-fed rat skeletal muscle was significantly lower (about 31%) than that in controls (P<0.01). Dietary treatment partly corrected fasting blood glucose [from(6.20±0.39)mmol/L to(5.78±0.74)mmol/L]and fasting serum insulin levels [from(17.19±1.93)mU/L to(11.68±1.28)mU/L] and increased the GLUT4 content at the plasma membrane by 1.14-fold in insulin-resistant rat skeletal muscle. CONCLUSION: High-fat feeding induces IR in Sprague-Dawley rats. The mechanism may be involved in decreased cell-surface level of GLUT4 through affecting intracellular insulin signaling and then decreasing GLUT4 trafficking.     

Effects of metformin combined with Ge Xia Zhu Yu decoction on TLR4/NF-κB signaling pathway in rats with polycystic ovary and insulin resis-tance

AIM: To study the effect of metformin (Met) combinated with Ge Xia Zhu Yu decoction on Toll-like receptor-4 (TLR-4)/nuclear factor-κB (NF-κB) signaling pathway in the rats with polycystic ovary syndrome (PCOS) and insulin resistance induced by dehydroepiandrosterone, and to explore the mechanisms. METHODS: PCOS rats (after induced by dehydroepiandrosterone, n=110) were randomly divided into 3 groups:model group (30 rats), Met treatment group (40 rats) and Met combinated with Ge Xia Zhu Yu decoction treatment (combination) group (40 rats). The rats in model group were given the same volume of 0.9% sodium chloride daily by gavage. The rats in Met group were given Met (270 mg·kg^-1·d^-1) by gavage. The rats in combination group were given Met (270 mg·kg^-1·d^-1) and Ge Xia Zhu Yu decoction (34.5 mg·kg^-1·d^-1) by gavage. All rats were continuously intervened for 28 d. After the intervention, blood glucose[fasting plasma glucose (FPG) and 2-hour postprandial blood glucose (2hPBG)] was measured. The mRNA expression levels of follicular epithelial NF-κB, TLR-4 and oxidized low-density lipoprotein (ox-LDL) were detected by RT-PCR. The serum levels of inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and C-reactive protein (CRP) were also detected by ELISA. RESULTS: After the intervention, FPG, 2hPBG, and serum levels of IL-6, TNF-α and CRP in Met group and combination group were lower than those in model group (P<0.05), and those in combination group were lower than those in Met group (P<0.05). Meanwhile, the mRNA expression levels of follicular epithelial NF-κB, TLR-4 and ox-LDL in Met group and combination group were lower than those in model group (P<0.05), and those in combination group were lower than those in Met group (P<0.05). CONCLUSION: Treatment with Met combined with Ge Xia Zhu Yu decoction improves insulin resistance in PCOS rats by decreasing the levels of inflammatory factors in serum and epithelial cells of ovary and inhibiting the expression of NF-κB, TLR-4 and ox-LDL in epithelial tissue of ovary.     

Effect of rosiglitazone on expressions of insulin receptor substrate-1 and glucose transporter 4 in skeletal muscle of type 2 diabetic rats with hyperlipemia

AIM: To investigate the effect of rosiglitazone on the expressions of insulin receptor substrate-1 (IRS-1) and glucose transporter 4 (GLUT4) in skeletal muscles of type 2 diabetic rats with hyperlipemia, and to explore the different pharmacological mechanism. METHODS: The model of type 2 diabetic rats with hyperlipemia was established by injecting low dosage of streptozotocin (STZ) and feeding with high fat diet. Then the diabetic rats were divided into two groups: untreated diabetic group and rosiglitazone-intervened diabetic group. The course of treatment lasted for 4 weeks. The expressions of IRS-1 and the GLUT4 proteins in the cell membrane of isolated rats skeletal muscles were detected by Western blotting. RESULTS: The fasting blood glucose, insulin and triglyceride contents in rosiglitazone-intervened diabetic group were lower than those in untreated diabetic group, but they were still higher than those in control group. The result of Western blotting showed that the expression of GLUT4 protein in rosiglitazone-intervened diabetic group was increased compared with untreated diabetic group, but its level was still lower than that in control group. The protein expression and tyrosine phosphorylation of IRS-1 in rosiglitazone-intervened diabetic group were significantly higher than those in untreated diabetic group and their levels were lower than those in control group. CONCLUSION: The effect of rosiglitazone on GLUT4 protein may link to its ability to induce the protein expression and tyrosine phosphorylation of IRS-1 in skeletal muscles in type 2 diabetic rats.     

Expression of IL-6 mRNA in endometrium with endometriosis

AIM: The purpose of the present study was to explore the relationship between interleukin-6 mRNA expression and endometriosis. METHODS: Using the rat model, IL-6 mRNA expression in the endometrium was examined by RT-PCR. RESULTS: The expression of IL-6 mRNA in control rats did not change at 2, 4, 6 and 8 weeks after sham operation (P>0.05), but in model rats it gradually increased at 2, 4, 6 and 8 weeks after endometriosis (P<0.01). The expression of IL-6 mRNA in uterine endometrium with endometriosis was lower than in endometriotic tissue, but higher than in endometrium from healthy controls. CONCLUSION: The IL-6 mRNA expression may contribute to the development of endometriosis . The increase in IL-6 mRNA expression may promote the implantation and growth of endometriotic tissue.     

The alteration of expression of iNOS mRNA and ecNOS mRNA in peripheral leukocytes from insulin resistance rats fed with fructose

AIM: To study the alteration of expression of iNOS mRNA and ecNOS mRNA in peripheral leukocytes of Wistar rats fed with fructose. METHODS: Wistar rats were randomly divided into the control group (n=10) and the fructose feeding group(n=10). The fructose feeding group drank12% fructose water for 6 months. The blood glucose, blood insulin, and the expression of iNOS mRNA and ecNOS mRNA in peripheral leukocytes of rats were determined. RESULTS: The levels of blood insulin (P<0.01) and the expression of ecNOS mRNA were higher in fructose feeding group than that in control group after1month. The level of blood insulin(P<0.01), the level of blood glucose (P<0.05), the expression of ecNOS mRNA and the iNOS mRNA were also higher in fructose feeding group than that in control group after 2 months. The levels of blood insulin and glucose, the expression of ecNOS mRNA and the iNOS mRNA were increased persistently during 3 to 6 months. CONCLUSIONS: These results indicate that fructose can increase the level, but reduce the sensitivity of insulin. It can also induce the expression of ecNOS mRNA firstly and the expression of iNOS mRNA secondly, the former can delay the formation of insulin resistance and the later can accelerate the formation of insulin resistance.     

Effect of microcapsulated catechin on expression of vascular endothelial growth factor in rats with adriamycin induced-nephrotic syndrome

AIM: To study the effect of microcapsulated catechin on vascular endothelial grower factor (VEGF) expression in rats with adriamycin induced-nephrotic syndrome.METHODS: 120 female SD rats were randomly distributed in control group,nephrotic group,dexamethason group,vitamin E group,catechin group and microcapsule group.Rat with nephrotic syndrome were induced by injection of adriblastine (5 mg/kg BW).VEGF concentrations in serum and urine were detected by ELISA assay.VEGF expression in kidney was measured by immunohistochemistry assay.RESULTS: At the end of 4th week and 6th week,VEGF concentration in other groups in kidney,serum and urine were higher than that in control (all P<0.01),and were lower than that in nephritic group (exclude Vit E group,all P<0.01).Serum and urine VEGF concentrations in microcapsule group were lower than those in catechin group (P<0.01,P<0.05,respectively).VEGF expression in microcapsule group in kidney was lower than that in catechin group,but it was not significant different.Urinary protein excretion at 24 h in microcapsule group was lower than that in catechin group (P<0.05),there was positive correlation among urine protein,serum VEGF level,urine VEGF concentration and renal VEGF expression at 24 h (all P<0.01).CONCLUSION: Catechin decreases urinary protein excretion in the rats with nephrotic syndrome through reducing the expression and secretion of VEGF.Microcapusulated catechin is benefit in decreasing the expression and secretion of VEGF.     

Post-receptor signaling crosstalk between GH and insulin in non-catch-up growth rats born small for gestational age

AIM:To investigate the post-receptor mechanism of growth hormone (GH) resistance and insulin (INS) resistance and their relationship in non-catch-up growth rats born small for gestational age (NCU-SGA), based on the post-receptor signalling cross-talk between GH and INS at PI3K signaling pathway. METHODS:NCU-SGA rat model was developed by food restriction to pregnant dams. 4 weeks old male NCU-SGA rats were studied. Total and phosphate insulin receptor substrate-1(IRS-1) and its downstream signal Akt levels in liver tissue were measured by Western blotting or immunoprecipitation at baseline, post-stimulating of insulin, and pre-treatment with JAK2 (post-receptor signaling protein of GH) inhibitor AG490 then given insulin stimulation, respectively. RESULTS:(1) Expression levels of total and phosphate IRS-1: No difference between NCU-SGA rats and normal control was observed (P>0.05). (2) Expression levels of Akt : At baseline, Akt was already activated in NCU-SGA rats compared to no Akt activation in normal control rats. However, post- stimulating of insulin, the increase level of phosphate Akt in NCU- SGA rats was remarkably lower than that in control rats (P<0.01). When pre-treatment with JAK2 inhibitor to block GH signaling pathway, the impaired Akt activity was significantly restored (P<0.01), which suggested that the signaling of GH uncouples signal transduction from IRS-1 to Akt in NCU-SGA rats.CONCLUSION:Insulin resistance is related to impaired IRS-1-Akt signaling pathway in NCU-SGA rats. GH resistance mediates and aggrevates INS resistance by uncoupling signal transduction from IRS-1 to Akt via signaling cross-talk at post-receptor level between GH and INS. PI3K/Akt may be the major site for this uncoupling.     

VEGF promotes biliary epithelial cell viability and cystic dilation in rats with polycystic kidney

AIM:To explore the promoting effect of vascular endothelial growth factor (VEGF) on the viability of biliary epithelial cells and biliary cystic dilation in rats with polycystic kidney (PCK). METHODS:Immunohistoche-mical staining was used to detect the expression of VEGF (n=6) and CD31 (n=10) in the liver tissue of normal and PCK rats. RT-qPCR and ELISA were used to evaluate the expression levels of VEGF in rat biliary epithelial cells and culture supernatant. WST-1 assay was applied to measure the effect of VEGF on the viability of rat biliary epithelial cells, and the influence of cholangiocyte culture supernatant on the viability of rat vascular endothelial cells. The cell migration assay was employed to observe the effect of cholangiocyte culture supernatant on endothelial cell migration. Tube formation assay was used to assess the impact of cholangiocyte culture supernatant on the angiogenic ability of endothelial cells. RESULTS:The result of immunohistochemical staining manifested that VEGF was highly expressed in the cholangiocytes of the PCK rats (P<0.01). More newly formed blood vessels were observed in the hepatic portal area of PCK rats than that in normal rats (P<0.01). The results of RT-qPCR and ELISA suggested that the mRNA and protein expression levels of VEGF in the cholangiocytes of PCK rats were significantly higher than those in normal rats (P<0.01). VEGF enhanced the viability of cholangiocytes in PCK rats (P<0.01). The culture supernatant of cholangiocytes in PCK rats increased the endothelial cell viability (P<0.01). VEGF siRNA and VEGF receptor inhibitor reduced the viability of cholangiocytes (P<0.01). The results of cell migration assay and tube formation assay indicated that the abilities of endothelial cell migration and tube formation were improved by the culture supernatant of cholangiocytes in PCK rats (P<0.01). CONCLUSION:The bile duct cystic dilation of PCK rats was related to the excessive secretion of VEGF in bile duct epithelial cells.     

Effects of low-glucose on long-term potentiation in the hippocampal slices of immature and adult rats

AIM: The effects of low-glucose on long-term potentiation (LTP) in the CA1 region of hippocapal slices of immature (15-16 days old) and adult (56-63 days old) rats were examined. METHODS: The technique of electrophysiology was used, and the slopes of the field excitatory postsynaptic potentials (S-EPSP) were measured. RESULTS: When slices were exposed to glucose medium at concentrations of 3 or 1.5 mmol/L, S-EPSP decreased significantly. In the slices from adult rats, only short-term potentiation was elicited by high frequency stimulation in the medium of 3 or 1.5 mmol/L glucose. However, in the slices from immature rats, LTP was still induced in the medium of 3 mmol/L glucose. CONCLUSION: Low-glucose medium depressed the synaptic transmission. In terms of the synaptic plasticity, the low-glucose endurance in immature rats was stronger than that in adult rats.     

Establishment and verification of insulin resistance model in mouse skeletal muscle cells

AIM:To establish a stable and repeatable insulin resistance model of skeletal muscle cells in vitro, so as to promote the exploration of the pathological mechanism of insulin resistance and the development and screening of related drugs. METHODS:C2C12 mouse myoblasts were used to induce differentiation in normal differentiation medium and differentiation medium containing glucose at 40 and 60 mmol/L, respectively. The effects of glucose at different concentrations on cell convergence, fusion and formation of multinucleated myotubes were observed under phase contrast microscope every day. After 1, 3, 5 and 7 d of differentiation, 2-NBDG assay was used to detect the effects of different interventions on C2C12 basal glucose uptake and insulin-stimulated glucose uptake. The effects of different interventions on the protein expression of glucose transporter 4 (GLUT4) after 5 d and 7 d of differentiation were determined by Western blot. The effects of different interventions on the distribution of GLUT4 protein after 5 d of differentiation were detected by immunofluorescence staining. RESULTS:After treated with glucose at 60 mmol/L, the morphological observation showed that high glucose treatment significantly inhibited the growth and differentiation of C2C12 cells after 3 d. High glucose treatment significantly inhibited basal glucose uptake and insulin-stimulated glucose uptake of the C2C12 cells after 5 d and 7 d (P<0.01). No difference between insulin-stimulated GLUT4 expression and basal GLUT4 expression after 5 d and 7 d of high glucose treatment was observed (P>0.05), but there was significant difference between control group and 60 mmol/L group (P<0.05) determined by Western blot. Immunofluorescence staining observation showed that the distribution of GLUT4 protein in the C2C12 cell membrane was significantly decreased after 5 d of high glucose treatment (P<0.01). Glucose treatment (40 mmol/L) also played a role to some extent, but the effect was not as obvious and stable as 60 mmol/L glucose. CONCLUSION:A stable insulin resistance model of mouse skeletal muscle cells in vitro was successfully established by high glucose stimulation. The treatment of glucose at 60 mmol/L for 5 d was the best. Morphological observation and detection of basic and insulin-stimulated glucose uptake and GLUT4 protein expression and distribution evaluates the insulin resistance level of skeletal muscle cells in vitro.     

Effect of aquaporin 4 on lymphatic brain edema in rats

AIM：To investigate the role of aquaporin 4 (AQP4) in the formation and elimination of lymphatic brain edema (LBE). METHODS：Sprague-Dawley rats were randomly divided into sham group and LBE group. LBE was induced by ligating bilateral cervical lymphatic tubes and removing the corresponding lymphatic nodes. The water contents in the cerebral cortex were measured by wet-dry weight method. The expression of AQP4 in the cerebral cortex was detected 1 d, 3 d, 7 d, 11 d and 15 d after a cervical lymphatic blockade or a sham operation by immunohistofluorescence staining and Western blotting. Furthermore, correlation analysis was made between the protein expression of AQP4 and the brain water content. RESULTS：Compared with sham group, cerebral water content, and the protein expression of AQP4 in the LBE rats increased significantly 3 d after operation, peaked at 7 d, followed by gradually stepping down. Furthermore, the expression of AQP4 was positively correlated with brain water content (r=0.8024, P<0.05). CONCLUSION：AQP4 may play an important role in the formation and elimination of LBE.     

Changes of IL-4, IL-10, IL-12 levels in rat serum and lung during acute respiratory distress syndrome induced by oleic acid

AIM:To observe the dynamic changes of interleukin-4(IL-4), IL-10, IL-12 in rat serum and lung tissues during acute respiratory distress syndrome (ARDS).METHODS:The ARDS model of rats was induced by intravenous injection of oleic acid. The levels of IL-4, IL-10, IL-12 in serum and the supernatant of lung tissues were measured by enzyme linked immunosorbent assay (ELISA).RESULTS:The Levels of serum and lung IL-10, IL-12 in ARDS rats were increased in 4 h, 8 h, 16 h group compared with control group. The levels in IL-10 in serum in 16 h group and IL-10 in lung tissues of 8 h group were lower than that in 4 h group. The Levels of IL-4 in serum in 4 h, 8 h group were higher than that in control group, while IL-4 in 16 h group was lower than that in 8 h group. IL-4 of lung tissues in 4 h, 8 h, 16 h group were increased significantly, but in 16 h group were lower than that in 8 h group. The biggest changes of pulmonary coefficient and histopathology were observed at 4 h after injection of oleic acid.CONCLUSIONS:IL-4, IL-10 and IL-12 might play important roles in inflammatory reaction induced by oleic acid. The pro-and anti-inflammatory cytokines produced successively during ARDS.The relationship between unbalanced cytokines and lung injury in ARDS needs to be further studied.     

Gene transfer and expression of recombined human proinsulin in hepatoma cells of rats

AIM:To explore the recombined human proinsulin gene containing glucose reaction element (GLRE) expression in transfected CBRH7919 cells. METHODS:The packaged retrovirus encoding genetically modified human proinsulin PLXSN-(GLRE)3-BP-1MpINS3 and PLXSN-(GLRE)3-BP-1MpINS2 were transfected into CBRH7919 cells. Insulin values in cells after transient and steady expression screened by G418 at different glucose levels were detected. Chromosome DNA was isolated from transfected and untransfected cells and polymerase chain reaction (PCR) was performed. PCR products were analyzed by electrophoresis. RESULTS:38 h after transfection, at the glucose levels of 0-25 mmol/L, the levels of insulin produced by cells including PLXSN-(GLRE)3-BP-1MpINS3 were (3.57±0.21)U/L, (5.30±0.20)U/L, (16.27±0.87)U/L, (23.23±1.12)U/L, respectively (P<0.05). One month after transfection, under above glucose levels, insulin values were (3.57±0.21)U/L, (5.30±0.20)U/L, (16.27±0.87)U/L, (23.23±1.12)U/L (P<0.05). CBRH7919 cells including PLXSN-(GLRE)3-BP-1MpINS2 secreted detectable insulin value at the level of 25 mmol/L, they were (2.10±0.23)U/L and (2.05±0.17)U/L, respectively. PCR products of transfected cells showed target band, but control cells did not. CONCLUSIONS:Recombined proinsulin gene was transfected successfully in CBRH7919 cells. The cells combined human proinsulin gene has the ability of producing insulin with increase in glucose concentration in vitro.