Objective To investigate the insulin receptor expression and binding characteristics of H22 rat hepatic cancer cell membrane with 125Iinsulin and the possibility of using insulin as a carrier for the receptor mediated targeted therapy. MethodsInsulin was radiolabelled using ChT method, isolated and purified by polyacrylamide gel electrophoresis, the labelled 125Iinsulin was measured by specific activity selfreplacement and over dose receptor combination experiment. The rat H22 hepatocarcinoma cells, normal rat hepatic cells were made, continuing the value Kd and Bmax of 125Iinsulin binding with insulin receptor on rat H22 hepatocarcinoma and normal hepatic cells membrane were evaluated in vitro, the competed binding curve was pictured. The binding Kd and Bmax value of 125Iinsulin receptor were evaluated with t test and receptor binding curve was tested with Scatchard method. ResultsThe Bmax value of rat H22 hepatocarcinoma cell receptor ［(5.6±1.1) pmol/106 cell］ was higher than that of normal hepatic cell ［(3.2±0.8) pmol/106 cell］ significantly (P＜0.05). The Kd value of H22 cell ［high and lowaffinity vs (1.8±0.6) nmol/L and (32.0±10.7) nmol/L］ and normal hepatic cell ［high and low affinity vs (2.1±0.9) nmol/L and (37.0±12.3) nmol/L］ were not significant respectively. In this experiment, it was specific when 125Iinsulin combined with receptor on H22 hepatocarcinoma cell and rat normal hepatic cell membrane. Conclusion This experiment showed that the receptor expresses much more significantly on H22 hepatocarcinoma cell membrane than that on normal rat hepatic cell membrane, 125Iinsulin combining with receptor on H22 hepatocarcinoma cell and rat hepatic cell membrane is highly specific. We may use insulin as an anticancer carrier to mediate insulin combined with receptor on hepatocarcinoma cell membrane in the target treatment of hepatic cancer.
Objective To investigate the ability of repairing bone defect with the compound of recombinant human insulinlike growth factor 1 (rhIGF-1), coralline hydroxyapatite(CHA) and autogeneous red bone marrow(ARBM), and to study the feasibility of the compounds being used as bone substitute materials. Methods Bilateral radius bone defects(11 mm in length) were created in 54 Chinese rabbits,which were randomly divided into 3 groups, and two different materials were randomly transplanted into the bilateral defects:in group 1, with material A(rhIGF-1/CHA/ARBM) and material B(CHA/ARBM); in group 2, with material C(rhIGF-1/CHA) and material D(CHA); in group 3, with E(autograft) and F(no implant) as controls. At 2, 4, 8 and 12 weeks, the effects were assessed by X-ray andimage analysis, biomechanics(at 12 weeks), as well as histological observation. Results X-ray and image analysis showed that material A of group 1was significantly superior to any other materials(P＜0.01). Antibending biomechanic detection showed that material A and Ewas significantly superior to the other materials(Plt;0.01), but no significant difference was found between A and E in the 12th week(Pgt;0.05). And by histological observation, in analogical bone morphological progress, materials C and D obviously inferior to materials A, B and E, but there was no significant difference between materials C and D. F had no evidence of new bone rebridging. Conclusion The recombinant compound CHA/ARBM(rhIGF-1),which posseses the potential ability of osteogenesis,osteoconduction and osteoinduction for bone defect repairing,can serve as a new type of autogenous bone substitute material.
Objective To study the therapeutic effect of Roux-en-Y gastric bypass （RYGB） on type 2 diabetes mellitus （T2DM） rats and explore the possible mechanism of vaspin in RYGB on T2DM. Methods Twenty SD rats with T2DM and 20 age- and sex-matched normal SD rats were randomly divided into 4 groups according to the random digits table：T2DM-RYGB group， T2DM-sham operation （SO） group，RYGB group，and SO group，10 rats in each group. Fasting plasma glucose （FPG） level，serum insulin （INS） level，vaspin level，and homeostasis model of insulin resistance （HOMA-IR） were determined before operation and on week 4，8 after operation，respectively．At the same time，the correlation between vaspin and the indicators （FPG，INS，or HOMA-IR） was analyzed．Results Compared the indicators after operation with before operation，the FPG level，INS level，vaspin level，and HOMA-IR were not significantly different between the T2DM-RYGB group and T2DM-SO group （P＞0.05） or between the RYGB group and SO group （P＞0.05），but the FPG level，INS level，vaspin level，and HOMA-IR in the T2DM-RYGB group and T2DM-SO group were significantly higher than those in the RYGB group （P＜0.05） and SO group （P＜0.05），respectively. On week 4 after operation，the FPG level，INS level，vaspin level，and HOMA-IR decreased in the T2DM-RYGB group，except for the FPG level，the other indexes had no significant differences as compared with the values before operation. On week 8 after operation，the FPG level，INS level，vaspin level，and HOMA-IR further decreased in the T2DM-RYGB group，there were significant differences of these indicators between before operation and on week 8 after operation. Compared the indicators after operation with before operation，the FPG level，INS level，vaspin level，and HOMA-IR were not statistically significant （P＞0.05） in the T2DM-SO group，RYGB group，or SO group. The changes in serum vaspin level correlated positively with those in INS and HOMA-IR before operaion and on week 4，8 after operaion in the T2DM-RYGB group and T2DM SO group rats （P＜0.05），respectively. Conclusions RYGB surgery has a therapeutic effect on T2DM rats，and serum vaspin level decreases and insulin resistance is improved after RYGB surgery，which may be one of the mechanisms of the treatment for T2DM.
ObjectiveTo explore the effect of endotoxin on insulin secretion from islet βcell of rat pancreas.MethodsAfter the model of endotoxemia was established in rats with intraperitoneal injection of LPS (2 mg/kg),the changes of insulin level in the serum and pancreas were dynamically determined, the expression of inducible nitric oxide synthase (iNOS) by situ hybridization and DNA damage in islet cells were also observed, the effect of sodium nitroprusside (exogenous NO) on synthesis and secretion of insulin from isolated islet βcell of normal rat pancreas under high glucose stimulation was also evaluated.ResultsThe level of glucose and insulin in plasma were significantly increased at 12th and 6th h, respectively and kept on 3 d after injection of LPS,but the insulin level in pancreas was not remarkably altered.The expression of iNOS and DNA damaged significantly enhanced at 6 d after endotoxemia. The high glucosestimulated insulin synthesis and secretion were bly inhibited by exogenous NO.ConclusionThese findings suggest that LPS be stimulate the expression of iNOS and NO product,which inhibites synthesis and secretion of insulin in islet βcells,but it stimulates insulin secretion by another mechanism,and results in dysfunction and destruction of the rat pancreas.
ObjectiveTo investigate role and mechanism of protein tyrosine phosphatase 1B (PTP1B) in jejunoileal bypass to treating rats with type 2 diabetes mellitus (T2DM).
MethodsTwenty-four T2DM SD rats and 24 normal SD rats were selected randomly by using random number table, then the SD rats with T2DM were randomly divided into jejunoileal bypass operation (DJBO, n=12) group and sham operation (DSO, n=12) group, the SD rats with normal food diet were randomly divided into jejunoileal bypass operation (NJBO, n=12) group and sham operation (NSO, n=12) group. Subsequently, fasting body weight (FBW), fasting plasma glucose (FPG), fasting insulin (FINS), and homeostasis model-insulin resistant (HOMA-IR) index of rats in each group were tested at different time points (before operation, on week 4 and 8 after operation). In addition, expression of PTP1B protein in skeletal muscle was determined by immunohistochemical staining and Western blot method respectively.
Results① The FBW before making T2DM model had no significant difference between the rats with high-fat diet and with normal diet (P > 0.05), which on week 4 or 8 after making T2DM model in the rats with high-fat diet was significantly heavier than that in the rats with normal diet (P < 0.05). ② Before jejunoileal bypass operation, the FBW, FPG, FINS, and HOMA-IR index in the DJBO group and the DSO group were significantly higher than those in the NJBO group and the NSO group (P < 0.05), respectively, which had no significant differences between the DJBO group and the DSO group (P > 0.05) and between the NJBO group and the NSO group (P > 0.05). ③ Compared with the values before jejunoileal bypass operation, the FBW, FPG, FINS, and HOMA-IR index on week 4 or 8 after jejunoileal bypass operation were significantly decreased in the DJBO group (P < 0.05); the FBW was significantly increased on week 4 or 8 after jejunoileal bypass operation in the DSO group and the NSO group (P < 0.05), and on week 8 after jejunoileal bypass operation in the NJBO group (P < 0.05). The other indexes had no significant differences between before and after jejunoileal bypass operation in the DSO group, the NSO group, or the NJBO group (P > 0.05). ④ On week 8 after jejunoileal bypass operation, the expression of PTP1B protein in the DSO group was significantly higher than that in the DJBO group, the NSO group or the NJBO group (P < 0.05), which in the DJBO group was significantly higher than that in the NSO group (P < 0.05) or the NJBO group (P < 0.05), which had no significant difference between the NJBO group and the NSO group (P > 0.05).
ConclusionJejunoileal bypass could effectively improve insulin resistance and decrease FPG level and FBW of T2DM rats through inhibiting expression of PTP1B protein in skeletal muscle of rat with T2DM.
Objective To investigate the relationship between the level of serum-insulin like growth factor-1( IGF-1) and the nut ritional status of cancerous cachexia. Methods Colon cancer CT-26 cells were implanted subcutaneously to 30 liver2specified IGF-1 gene deleted (L ID) C57BL/ 6 mice to establish cancerous cachexia model and theother 30 C57BL/ 6 mice were included as cont rol group. The serum levels of IGF-1 , cytokine TNF-αand IL-6 , bloodglucose , albumin and t riglyceride were detected respectively on day 14 , 18 and 22 af ter the plantation of tumor. Thebody weight of mice , tumor weight and the weight af ter tumor removed in two group s were measured respectively.Results Af ter the plantation , the levels of IGF-1 in L ID group at different times were all significantly lower thanthose in cont rol group ( Plt; 0. 05) . The serum levels of TNF-α, IL-6 , blood glucose and t riglyceride were ascendinggradually over time ( Plt; 0. 05) , but weight s af ter tumor removed and the level of albumin were descending in twogroup s ( Plt; 0. 05) . Compared with the cont rol group , the serum levels of IL-6 , TNF-α, blood glucose and t riglyceride in L ID tumor-bearing mice were all significantly higher at different time point s ( P lt; 0. 05) . On day 18 and 22 ,the weight s af ter tumor removed and the amount of ingestion in L ID group were significantly lower than those in thecont rol group ( Plt; 0. 05) . Conclusion Compared with the low level of IGF-1 in cancerous cachexia , normal level ofserum IGF-1 may represent lower degree of cancerous cachexia2related cytokines and better nut ritional state , whichmay provide a novel idea of the therapy of cancerous cachexia.
OBJECTIVE: To investigate the effect of electroacupuncture on mRNA expression of NGF and IGF-1 in injured nerve. METHODS: Sciatic nerve injury model was established by transection of right side sciatic nerve in 90 male SD rats, which were randomly divided into two groups. The experimental group was treated with electroacupuncture, no treatment in the control group. The distal part of the injured nerve was harvested after 1, 2, 4, 6 and 10 weeks of operation and stored in the liquid nitrogen. The total RNA was extracted by the TRIzol reagent. Reverse transcriptase-polymerase chain reaction(RT-PCR) was used to detected the mRNA expression of NGF and IGF-1. RESULTS: The mRNA expression of NGF in the experimental group was increased quickly from the second week, and reached to highest level in the fourth week. It was much higher than that of the control group (P lt; 0.05). Then it began to decline in following time and approximately reached to the level of the first week after 10 weeks of operation. The mRNA expression of IGF-1 in the experimental group was remarkably increased in the second and fourth week, and which was much higher than that of the control group respectively(P lt; 0.05). Although the mRNA expression of IGF-1 after 10 weeks of operation in the experimental group was higher than that of the control group, but there was no significant difference between the two groups(P gt; 0.05). There was linear correlation in the fourth week between mRNA expression of NGF and IGF-1 in the experimental group. CONCLUSION: The mRNA expression of NGF and IGF-1 can be elevated in injured nerve at early stage interfered with electroacupuncture.
Objective To investigate the effect of combined delivery of hepatocyte growth factor (HGF) and insulinlike growth factor-1 (IGF-1) on the development of bone mesenchymal stem cells (BMSCs) differentiation by expression of GATA-4，and to supply some evidence for clinical BMSCs transplantation therapy. Methods BMSCs were isolated from the femurs and tibias of the randomly assigned rabbits and cocultured with myocytes in a ratio of 1∶1. Myocytes were obtained from neonatal rabbits ventricles. 150 ng/ml HGF and 200 ng/ml IGF-1 were added into 4 culture bottles of 8 bottles and the other 4 bottles were not. After BMSCs were cocultured with myocytes for 1 day, 3 days, 1 week, and till 6 weeks， differentiated BMSCs were targeted and microdissected with a laser capture microdissection system, and then ribonucleic acid (RNA) was extracted and isolated. The differentiation of BMSCs in coculture was confirmed by immunohistochemistry, electron microscopy, and reverse transcriptionpolymerase chain reaction (RT-PCR). And expression of GATA-4 in BMSCs was detected by semiquantitative RT-PCR. Results Before coculturing, the BMSCs were negative for α-actinin and exhibited a nucleus with many nucleoli. After coculture with myocytes, some BMSCs became αactininpositive and showed a cardiomyocytelike ultrastructure, including sarcomeres, endoplasmic reticulum, and mitochondria. BMSCs cocultured with myocytes expressed cardiac transcription factor GATA-4. IGF-1 and HGF delivery can significantly increased expression of GATA-4 for the differentiated BMSCs as compared with cells of no delivery of HGF and IGF-1. The expression level of GATA-4 in captured BMSCs began to increase at the 1st day, reach the peak at the 2nd week and kept high expression level after the 2nd week. Conclusion BMSCs can transdifferentiate into cells with a cardiac phenotype when cocultured with myocytes. Differentiated myocytes express cardiac transcription factors GATA-4. Administration of HGF and IGF-1 promoted the development of BMSCs transdifferentiate into cardiac phenotype, which is associated with the increase in expression level of GATA-4.
ObjectiveTo evaluate the prognostic significance of serum levels of vascular endothelial growth factor (VEGF) and insulin-like growth factors-Ⅰ (IGF-Ⅰ) in advanced gastric cancer patients who were treated with oxaliplatin/5-fluorouracil (FOLFOX).
MethodsNinety-six advanced gastric cancer patients who were treated with FOLFOX in our hospital between March 2007 to August 2010 were enrolled in this study. All of the patients were treated with oxaliplatin (85 mg/m2) as a 2-hour infusion on day 1, and leucovorin (20 mg/m2, about 10 min) on day 1 and day 2, followed by a 5-fluorouracil bolus (400 mg/m2) and 22 hours of continuous infusion of 600 mg/m2. Treatment was repeated in 2-week intervals, and patients received 4 chemotherapy cycle in total. The levels of serum VEGF and IGF-Ⅰ were measured using enzyme-linked immunoassays. The relationship between serum levels of VEGF/IGF-Ⅰ and the clinicopathological characteristics of patients, the relationship between serum levels of VEGF/IGF-Ⅰ and prognosis of patients, were analyzed.
ResultsThe serum levels of VEGF and IGF-Ⅰ were (464.4±57.4) pg/mL and (33.5±7.3) ng/mL, respectively. The serum level of VEGF was related with surgical history, Lauren's classification, TNM staging before treatment, and pathological type (P < 0.05), and serum level of IGF-Ⅰ was related with TNM staging before treatment and number of transferred organs (P < 0.05). The serum levels of VEGF and IGF-Ⅰ in stable disease (SD) +progressive disease (PD) patiens were higher than those of complete response (CR) +partial response (PR) patients (P < 0.05). The results of Cox proportional hazard regression model showed that, effect of chemotherapy (HR=1.764, P=0.006), number of transferred organs (HR=1.662, P=0.015), serum level of VEGF (HR=1.834, P=0.012) and IGF-Ⅰ (HR=1.855, P=0.008), were all significantly related with time to progression (TTP); serum level of VEGF (HR=2.205, P=0.002) and IGF-Ⅰ (HR=1.931, P=0.004) were all significantly related with overall survival (OS).
ConclusionLevels of serum VEGF and IGF-Ⅰ are independent prognostic factors in patients with advanced gastric cancer who were treated with FOLFOX chemotherapy.
【Abstract】 Objective To explore the new therapy for pulmonary fibrosis by observing the effects of insulin-like growth factor 1 ( IGF-1) treated mesenchymal stemcells ( MSCs) in rats with bleomycin-induced pulmonary fibrosis. Methods Bone marrowmesenchymal stemcells ( BMSCs) were harvested from6-week old male SD rats and cultured in vitro for the experiment. 48 SD rats were randomly divided into 4 groups, ie.a negative control group ( N) , a positive control group/bleomycin group ( B) , a MSCs grafting group ( M) ,and an IGF-1 treated MSCs grafting group ( I) . The rats in group B, M and I were intratracheally injected with bleomycin ( 1 mL,5 mg/kg) to induce pulmonary fibrosis. Group N were given saline as control. Group M/ I were injected the suspension of the CM-Dil labled-MSCs ( with no treatment/pre-incubated with IGF-1 for 48 hours) ( 0. 5mL,2 ×106 ) via the tail vein 2 days after injected bleomycin, and group B were injected with saline ( 0. 5 mL) simultaneously. The rats were sacrificed at 7,14,28 days after modeling. The histological changes of lung tissue were studied by HE and Masson’s trichrome staining. Hydroxyproline level in lung tissue was measured by digestion method. Frozen sections were made to observe the distribution of BMSCs in lung tissue, and the mRNA expression of hepatocyte growth factor ( HGF) was assayed by RTPCR.Results It was found that the red fluorescence of BMSCs existed in group M and I under the microscope and the integrated of optical density ( IOD) of group I was higher than that of group M at any time point. But the fluorescence was attenuated both in group M and group I until day 28. In the earlier period, the alveolitis in group B was more severe than that in the two cells-grafting groups in which group I was obviously milder. But there was no significant difference among group I, M and group N on day 28.Pulmonary fibrosis in group B, Mand I was significantly more severe than that in group N on day 14, but itwas milder in group M and I than that in group B on day 28. Otherwise, no difference existed between the two cells-grafting groups all the time. The content of hydroxyproline in group B was significantly higher than that in the other three groups all through the experiment, while there was on significant difference betweengroup I and group N fromthe beginning to the end. The value of group M was higher than those of group I and group N in the earlier period but decreased to the level of negative control group on day 28. Content of HGF mRNA in group Nand group I was maintained at a low level during the whole experiment process. The expression of HGF mRNA in group I was comparable to group M on day 7 and exceeded on day 14, the difference of which was more remarkable on day 28. Conclusions IGF-1 can enhance the migratory capacity of MSCs which may be a more effective treatment of lung disease. The mechanismmight be relatedto the increasing expression of HGF in MSCs.
