3T3L1脂肪细胞对大鼠胰岛β细胞功能障碍的影响

Objective To investigate the integrated effects of adipocytes on rat beta-cells, differentiated 3T3L1 adipocytes and rat islet cells co-culture system was established. Methods There were two groups; control group ( SD rat islet cells) and co-culture group (islet cells and 3T3L1 adipocytes co-culture system). Islet cells were obtained for determination of (1) insulin secretion and insulin content; (2) mRNA expressions of GLUT2, GCK and Kir6. 2; (3) protein expressions of IR-β, IRS-1 and their tyrosine phosphorylation leveL Results (1) At low glucose, insulin secretion of co-culture group increased compared with that of control group (0.79±0.35) ng·h-1 · ml-1 islet vs (0.38±0.09 ) ng · h-1 · ml-1 · islet, P = 0. 028 . At high glucose, insulin secretion of those two groups was almost at the same level (P =0.760). Compared with control group (2. 84 ±0.92) , stimulation index (SI, insulin release at high glucose/ low glucose) of co-culture system decreased to (1. 57 ±0. 61, P =0.04). And the insulin content of the both groups was almost at the same level (P=0.102). (2) The mRNA of GCK, GLUT2 and Kir6. 2 in co-culture group downregulated to (0. 27 ±0. 11, P = 0. 01) , (0. 34 ±0. 24, P = 0. 009) and (0.41 ± 0. 09, P = 0. 003) compared with control group (mRNA = 1). ( 3) The protein levels of IR-β, IRS-1 and their tyrosine phosphorylation decreased in co-culture system. Conclusions 3T3L1 adipocytes are involved in beta-cell dysfunction, which may facilitate the development of type 2 diabetes. The effects may be mediated by multiple pathways, which include downregulation of GSIS related gene expressions and suppression of islet cell insulin signaling.     

腺病毒介导的白细胞介素10基因转移对胰岛β细胞凋亡及胰岛素释放的影响

Objective To evaluate the efficacy of adenovirus vector-mediated murine interleukin-10 (mIL-10) gene transfer to rat beta cell-RINm5F cells in vitro and to explore the potential value of gene therapy in type 1 diabetes mellitus. Methods The recombinant adenovirus vector Ad-mIL-10 was constructed and transfected into RINm5F cells (Ad-mIL-10 group). Untransfected RINm5F cells and Ad-eGFP-transfected cells were used as controls. The expression of mIL-10 was examined by RT-PCR and Western blot The levels of IL-10 in supernatant were measured by enzyme-linked immunosorbent assay (ELISA). For a determination of insulin release, the cells were cultured with high glucose ( 16. 7mmol/L) for a 1 -hour co-incubation. Then radioimmunoassay was used to detect the level of insulin in supernatant After induction of IL-1β, the levels of nitric oxide (NO) and nitric oxide synthase ( NOS) were measured, the apoptosis of transfected cells was detected by Hoechst 33258 staining and Fas expression by flow cytometry. Results Both mRNA and protein of Ad-mIL-10 were successfully expressed in RINm5F cells. Expression of mIL-10 gene resulted in significant increases in insulin secretion in response to high glucose. Compared with uninfected control and Ad-eGFP infected group, Ad-mIL-10 infected group had decreased levels of NO and NOS induced by IL-lβ [ NO level ( nmol/106 cells): 52. 9 ± 3. 2 vs 227. 3 ± 26. 4, 235. 1 ±28.6, both P＜0.05; NOS level (U/106 cells): 9. 3 ±1.2 vs 29. 8 ±2.5, 30.5 ±2.8, both P ＜ 0. 05]. Furthermore, Ad-mIL-10 gene transfer led to a profound reduction of Fas-expressing islet cells under the induction of IL-1β. Fas-expressing islet cells in Ad-mIL-10 group were significantly lower than those in uninfected group and Ad-eGFP-transfected group (24. 6% ± 1.0% vs 33. 3% ± 5. 1% , 32. 6% ±1.1%) (P＜0.05).The apoptotic rates of Ad-mIL-10 group were lower in comparison with the other two groups (9.4%±1.1% vs 19.2%±2.2%,20.6%±2.3%,P＜0.05).Conclusions IL-10 gene transfer to islet beta cells may be beneficial in maintaining beta cells function,protecting islet cells from IL-1β-mediated apoptosis and promoting islet cells survival.It is a potential therapy for type 1 diabetes mellitus.     

腺病毒介导的白细胞介素10基因转移对胰岛β细胞凋亡及胰岛素释放的影响

Objective To evaluate the efficacy of adenovirus vector-mediated murine interleukin-10 (mIL-10) gene transfer to rat beta cell-RINm5F cells in vitro and to explore the potential value of gene therapy in type 1 diabetes mellitus. Methods The recombinant adenovirus vector Ad-mIL-10 was constructed and transfected into RINm5F cells (Ad-mIL-10 group). Untransfected RINm5F cells and Ad-eGFP-transfected cells were used as controls. The expression of mIL-10 was examined by RT-PCR and Western blot The levels of IL-10 in supernatant were measured by enzyme-linked immunosorbent assay (ELISA). For a determination of insulin release, the cells were cultured with high glucose ( 16. 7mmol/L) for a 1 -hour co-incubation. Then radioimmunoassay was used to detect the level of insulin in supernatant After induction of IL-1β, the levels of nitric oxide (NO) and nitric oxide synthase ( NOS) were measured, the apoptosis of transfected cells was detected by Hoechst 33258 staining and Fas expression by flow cytometry. Results Both mRNA and protein of Ad-mIL-10 were successfully expressed in RINm5F cells. Expression of mIL-10 gene resulted in significant increases in insulin secretion in response to high glucose. Compared with uninfected control and Ad-eGFP infected group, Ad-mIL-10 infected group had decreased levels of NO and NOS induced by IL-lβ [ NO level ( nmol/106 cells): 52. 9 ± 3. 2 vs 227. 3 ± 26. 4, 235. 1 ±28.6, both P＜0.05; NOS level (U/106 cells): 9. 3 ±1.2 vs 29. 8 ±2.5, 30.5 ±2.8, both P ＜ 0. 05]. Furthermore, Ad-mIL-10 gene transfer led to a profound reduction of Fas-expressing islet cells under the induction of IL-1β. Fas-expressing islet cells in Ad-mIL-10 group were significantly lower than those in uninfected group and Ad-eGFP-transfected group (24. 6% ± 1.0% vs 33. 3% ± 5. 1% , 32. 6% ±1.1%) (P＜0.05).The apoptotic rates of Ad-mIL-10 group were lower in comparison with the other two groups (9.4%±1.1% vs 19.2%±2.2%,20.6%±2.3%,P＜0.05).Conclusions IL-10 gene transfer to islet beta cells may be beneficial in maintaining beta cells function,protecting islet cells from IL-1β-mediated apoptosis and promoting islet cells survival.It is a potential therapy for type 1 diabetes mellitus.     

Effects of L-lysine monohydrochloride on insulin and blood glucose levels in spinal cord injured rats

Background Hyperglycemia in brain and spinal cord could aggravate neurologic impairment. Recent studies showed that L-lysine monohydrochlonde （LMH） could increase the insulin secretion and regulate the blood glucose level. The aim of the present study was to investigate the effects of LMH on pancreatic islet B cells, the levels of endogenous insulin and blood glucose in spinal cord injured rats.Methods Forty male Wistar rats were divided into four groups, namely, normal control group, model group, high-dose LMH group （621.5 mg/kg equal to LMH 1/8 LD50）, and low-dose LMH group （310.8 mg/kg equal to LMH 1/16 LD50）. The model of spinal cord injured rat was established by hemi-transection at the lower right thoracic spinal cord. LMH was administered via intraperitoneal injection once spinal cord injury was produced in rats. All rats were sacrificed 48 hours after spinal cord injured. The effects of LMH on pancreatic islet B cells, the content of endogenous insulin, end the level of blood glucose were observed with immunohistochemical method, radioimmunoassay method, end biochemical analyzer, respectively. Results The insulin immunohistochemical intensities of islet B cells were significantly weaker in model group then those in normal control group （P 〈0.01）. The levels of endogenous insulin were significantly lower and the blood glucose levels were significantly higher in model group than those in normal control group （P 〈0.01）. The insulin immunohistochemical intensities of islet B cells were significantly stronger in high-dose LMH group then those in model group （P〈0.05）. In addition, we found that the levels of endogenous insulin were significantly higher and the blood glucose levels were significantly lower in high-dose LMH group then those in model group （P 〈0.05）. There were no significant differences in the insulin immunohistochemical intensities of islet B cells, the levels of endogenous insulin and the blood glucose between low-dose LMH group and model group （P 〉0.05）. Conclusion LMH, but dose-dependent, might participate in the regulation of pancreatic islet B cells, and then reduce the blood glucose levels in the spinal cord injured rats.     

Protection of human islets from induction of apoptosis and improved islet function with HO-1 gene transduction

Background Islet transplantation represents an ideal therapeutic approach for treatment of type 1 diabetes but islet function and regeneration may be influenced by necrosis or apoptosis induced by oxidative stress and other insults. Heme oxygenase-1 (HO-1) is the rate-limiting enzyme in the catabolism of heme into biliverdin, releasing free iron and carbon monoxide. It has also been reported to be an antioxidant enzyme which can improve the function of grafted islets by cytoprotection via free radical scavenging and apoptosis prevention. In the present study, we investigated whether transduction of HO-1 genes into human islets with an adenovirus vector has cytoprotective action on islets cultured in vitro and discuss this method of gene therapy for clinical islet transplantation.Methods Cadaveric pancreatic islets were isolated and purified in vitro. Transduction efficiency of islets was determined by infecting islets with adenovirus vector containing the enhanced green fluorescent protein gene (Ad-EGFP) at multiplicities of infection (MOI) of 2, 5, 10, or 20. Newly isolated islets were divided into three groups: EGFP group, islets transduced with Ad-EGFP using MOI＝20; HO-1 group, transduced with adenovirus vectors containing the human HO-1 gene using MOI＝20; and control group, mock transduced islets. Insulin release after glucose stimulation of the cell lines was determined by a radioimmunoassay kit and the stimulation index was calculated. Flow cytometry was used to detect apoptotic cells in the HO-1 group and in the control group after induction by recombinant human tumor necrosis factor-α (rTNFα) and cycloheximide (CHX) for 48 hours.Results Adenovirus vectors have a high efficiency of gene transduction into adult islet cells. Transduction of islets with the Ad-EGFP was most successful at MOI 20, at which MOI fluorescence was very intense on day 7 after transduction and EGFP was expressed in cultured islet cells for more than four weeks in vitro. The insulin release in the control group was (182.36±58.96) mIU/L after stimulation by high glucose media (16.7 mmol/L), while insulin release from the HO-1 group and the EGFP group were (270.09±89.37) mIU/L and (175.95±75.05) mIU/L respectively. Compared to the control group and the EGFP group, insulin release in the HO-1 group increased significantly (P＜0.05). After treatment with rTNFα and CHX the apoptotic ratio of islet cells was (63.09±10.86)% in the HO-1 group, significantly lower than (90.86±11.25)% in the control group (P<0.05).Conclusions Transduction of human islets with Ad-HO-1 can protect against TNF-α and CHX mediated cytotoxicity. The HO-1 gene also appears to facilitate insulin release from human islets. Transduction of donor islets with the adenovirus vector containing an HO-1 gene might have potential value in clinical islet transplantation.     

Insulin Resistance and Hypertension

The insulin sensitivity in hypertensive patients with normal glucose tolerance (NGT),impaired glucose tolerance (IGT) and type 2 diabetes mellitus (DM) and the insulin resistance (IR) under the disorder or grucose metabolism and hypertension were studied.by glucose tolerance test and insulin release test,insulin sensitivity index (ISI) and the ratio of area under glucose tolerance curve (AUCG) to area under insulin release curve (AUC1) were calculated and analyzed.The results showed that ISI was decreased to varying degrees in the patients with hypertension,the mildest in the group of NGT with hypertension,followed by the group of IGT without hypertension,the group of IGT with hypertension and DM(P=0).There was very significant difference in the ratio of AUCG/AUC1 between the hypertensive patients with NGT and controls (P=0).It was concluded that a significant IR existed during the development of IGT both in hypertension and nonhypertension.The increase of total insulin secretion (AUC1) was associated with nonhypertension simultaneously.IR of the hypertensive patients even existed in NGT and was worsened with the deterioration of glucose metabolism disorder,but the AUC1 in the HT group changed slightly.A relative deficiency of insulin secretion of dysfunction of β-cell of islet existed in IGT and DM of the hypertensive patients.     

Effects of High Concentration Glucose on the Expression of NF-κB, Bax and Cytochrome C and Apoptosis of Islet Cells in Mice

The roles of NF-kappaB （NF-κB） expression, Bax activity and cytochrome C （Cyt C） release, apoptosis of islet cells induced by high concentration glucose were explored in vitro. Pancreatic islet cells, which were isolated from Kunming mice, were cultured with different concentrations of glucose in DMEM, and divided into the following groups： G1, G2, G3, G4, G5, and G6 groups, corresponding to the glucose concentrations of 5.6, 7.8, 11.1, 16.7, 22.5, and 27.6 mmol/L, respectively. After culture for 120 h, insulin secretion was evaluated by radioimmunoassay, and the NF-rd3 expression was detected by immunocytochemistry. Bax activity and Cyt C release were measured by immunofluorescence, and apoptosis was examined by Hoechst33342 assay. The results showed that in GI, G2 and G3 groups, insulin secretion was enhanced with the increase of glucose concentration, and the NF-κB expression was also increased （P〈0.05）, but Bax activity, Cyt C release and apoptosis rate showed no significant difference among them. However, in G4, G5, and G6 groups, apoptosis rate of islet cells, NF-rd3 expression, Bax activity, and Cyt C release were all significantly increased, and insulin secretion was impaired as compared with G1, G2, and G3 groups （P〈0.05）. It was concluded that the exposure of islet cells to high glucose could induce islet cells apoptosis as well as impaired insulin secretion. The NF-κB signaling pathway and mitochondria pathway in islet cells might play some roles in the progressive loss of islet cells in diabetes. The inhibition of the NF-κB expression could be an effective strategy for protecting pancreatic islet cells.     

Protection of rat islet viability following heme oxygenase-1 gene transfection via adenoviral vector in vitro

Objective： To investigate the effect of Heme oxygenase-1 （HO-1） gene transfection on the viability of cultured rat islets, and to explore the potential value of HO-1 gene in islet transplantation. Methods：Recombinant adenovirus vector containing human HO-1 gene（Ad-HO-1 ） or enhanced green fluorescent protein gene（Ad-EGFP） was generated by using AdEasy system respectively. The rat islets were transfected with Ad-HO-1, Ad-EGFP or blank vector and then cultured for 7 days. Transfection was confirmed by expression of EGFP and human HO-1 protein detected by fluorescence photographs and western blot, respectively. The insulin release upon different concentration of glucose stimulation was detected using insulin radioimmunoassay kit, and stimulation index（SI） was calculated. Glucose-stimulated insulin release was used ＇to assess islet viability. Results：Adenovirus vector successfully transferred HO-1 gene to rat islet cells in vitro, and the insulin release upon high level of glucose stimulation and stimulation index （SI） of Ad-HO-1-infected islets were significantly higher than those of Ad-EGFP-infected islets and control islets （P 〈 0.05）. Conclusion： Adenovirus-mediated HO-1 gene transfection is a feasible strategy to confer cytoprotection and therefore protect the viability of cultured rat islets.     

力达霉素对神经胶质瘤细胞血管生成拟态的抑制及对细胞凋亡的影响

Objective To evaluate the in vitro effects of lidamycin upon vasculogenic mimicry and apoptosis induction in glioma cells. Methods Tube formation assay was performed to estimate the inhibitory effects of lidamycin upon vasculogenic mimicry in C6 and U87 glioma cells. The vasculogenic mimicry of glioma cells was photographed and enumerated. Annexin V-FITC/PI was used for determination of glioma cell apoptosis with flow cytometry. Results Vasculogenic mimicry assay indicated that 0. 1 nmol/L, 0. 5 nmol/L and 1 nmol/L lidamycin showed significant inhibition of vasculogenic mimicry in C6 and U87 cells. Comparing with C6 control group (14. 7 ± 1.2), 0. 1 nmol/L (12. 7 ± 0. 6), 0. 5 nmol/L (9. 0 ± 1.7) and 1 nmol/L (4. 7 ± 0. 6) lidamycin inhibited vasculogenic mimicry in C6 cells with statistical significances (P = 0. 013, P = 0. 005 and P = 0. 0002 respectively). Comparing with U87 control group (14.7±1.2), the vasculogenic mimicry of 0.1 nmol/L (10.0±2.0), 0.5 nmol/L (8.3±1.5) and 1 nmol/L lidamycin (4. 3±0. 6) treated U87 cells showed statistical significances (P =0. 025, P =0. 005 and P =0. 0009 respectively). The apoptotic ratios of same dosages lidamycin treated C6 cells and U87 cells showed a similar tendency as vaaculogenic mimicry inhibition (P < 0. 001). Lidamycin was more potent than neocarzinostatin in vasculogenic mimicry inhibition and apoptosis induction of C6 cells and U87 cells. Conclusion Lidamycin can inhibit vasculogenic mimicry and promote apotosis of glioma cells. Thus it is a promising drug in glioma treatment. Further researches on the therapeutic efficacy of enediyne antibiotics in glioma are needed.     

14—3—3蛋白

14-3-3是一个在真核生物中广泛表达的酸性蛋白家族，主要以同源／异源二聚体形式存在，通过磷酸化丝／苏氨酸作用与靶蛋白或靶蛋白的两个结构域结合。七种不同亚型的高度保守的14-3-3蛋白与人类细胞中多种不同的信号通路有着密切的关系。14-3-3通过调节靶蛋白间的相互作用在MAPK级联放大效应等信号转导途径中发挥调控作用。     

微卡（VACCAE）联合2H3R3Z3E3/4H3R3方案治疗初治涂阳肺结核的临床效果

目的应用微卡(VACCAE)联合常规抗结核药物2H3R3Z3E3/4H3R3治疗初治涂阳肺结核的临床疗效。方法选取100例初始涂阳肺结核患者,随机分为对照组50例和观察组50例,两组均采用"世界银行贷款+中国结核病控制项目"的2H3R3Z3E3/4H3R3方案治疗,观察组在上述常规化疗药基础上联用微卡22.50μg深部肌肉注射;观察两组的痰菌阴转率、病灶吸收率、空洞闭合率、临床症状改善情况、不良反应发生率。结果治疗1个月后,观察组临床症状完全消失率显著高于对照组(P<0.05)。观察组的痰菌转阴率、病灶吸收率、空洞闭合率均优于对照组(P<0.05)。微卡注射后无严重不良反应。结论微卡联合常规抗结核药物2H3R3Z3E3/4H3R3方案治疗初治涂阳肺结核能够快速缓解临床症状,且疗效显著不良反应小,具有临床应用价值。     

Hypoxia-induced MTA1 promotes MC3T3 osteoblast growth but suppresses MC3T3 osteoblast differentiation

Background

Bone fracture is one of the most common physical injuries in which gene expression and the microenvironment are reprogramed to facilitate the recovery process.

Methods

By specific siRNA transfection and MTT assay, we evaluated the effects of metastasis-associated gene 1 (MTA1) in osteoblast growth. To show the role of MTA1 in osteoblast under hypoxia conditions, by overexpressing and silencing MTA1 expression, we performed mineral deposition and alkaline phosphatase activity assay to observe the differentiation status of osteoblast cells. Real-time PCR and Western blot assays were adopted to detect the expression of certain target genes.

Results

Here, we reported that hypoxia-induced MTA1 expression through hypoxia-induced factor 1 alpha (HIF-1α) and stimulated the growth of osteoblast MC3T3 cells. Silencing of MTA1 through specific siRNA suppressed MC3T3 cell growth and elicited cell differentiation and induced alkaline phosphatase activation and the upregulation of bone morphogenetic protein-2 and osteocalcin.

Conclusions

We found that MTA1 was regulated by HIF-1α in hypoxia circumstance to suppress osteoblast differentiation. These findings provide new insights for bone fracture healing and new strategies to develop potential targets to promote fracture healing.

Electronic supplementary material

The online version of this article (doi:10.1186/s40001-015-0084-x) contains supplementary material, which is available to authorized users.     

稳定表达神经营养素-3的NIH3T3细胞建立及鉴定

目的：建立稳定表达NT3的NIH3T3细胞株，通过体外共培养实验观察其对耳蜗螺旋神经节细胞生长的影响．方法：以阳离子脂质体作为载体，将携带有外源性基因NT3的重组真核表达载体pIRES2-EGFP-NT3转染小鼠成纤维细胞，进行RT-PCR，Western-blot分析及免疫组化鉴定，并与耳蜗螺旋神经节细胞进行共培养实验．结果：得到抗G-418的阳性细胞克隆；RT-PCR的产物约为744 bp；Western-blot可见一清晰的蛋白条带，与NT3的蛋白分子量相符合．NT3免疫组化染色结果显示转染NT3-cDNA的NIH3T3细胞胞质呈棕黄色着色．NT3转染组细胞与耳蜗螺旋神经节细胞共培养2wk后，与对照组相比，耳蜗螺旋神经节细胞无明显减少．结论：成功建立了稳定表达NT3的NIH3T3细胞株；该细胞对耳蜗螺旋神经节细胞有很好的营养支持作用．     

EB病毒核抗原3C提高Gemin3基因的表达

  目的 探讨EB病毒核抗原(EBNA)3C对Gemin3基因表达的影响。方法 共转染EBNA3C和Gemin3基因至HEK293细胞。依靠慢病毒载体介导发卡RNA干涉敲减EBNA3C基因的表达,并经嘌呤霉素筛选获得稳定的EBNA3C低表达细胞系,采用Western blot检测EBNA3C对Gemin3蛋白表达的影响。结果 Gemin3基因的表达随着EBNA3C表达量的增加而增加,呈剂量依赖性,在EBNA3C基因敲减细胞中Gemin3的表达减少。结论EBNA3C可提高Gemin3基因表达水平。     

新生儿脐血血清rT_3及T_3/rT_3值的测定

测定300例新生儿脐血血清T_4、T_3、rT_3及T_3/rT_3值。其平均值±标准差依次为98.65±27.6ug/L,0.49±0.19ug/L,3.06±0.25ug/L;T_3/rT_3值平均为0.16。脐血T_4、rT_3值高于正常成人,而T_3及T_3/rT_3值明显低于正常成人。     

血小板生长因子在NIH3T3成纤维细胞和MT3转化细胞应答中的作用

血小板生长因子(PDGF)通过促进细胞外Ca~(2 )内流,增加细胞内Ca~(2 )浓度,但在亲代NIH3T3成纤维细胞和转化的MT3细胞中,却表现出不同的作用。在亲代NIH3T3成纤维细胞中,PDGF能够抑制舒缓激肤介导的细胞内Ca~(2 )浓度增加,未能见到细胞内Ca~(2 )波动现象;而在转化的MT3细胞中,PDGF丧失了对舒缓激肽介导的细胞内Ca~(2 )浓度增加的抑制作用,常常引起细胞内Ca~(2 )波动现象。对于DNA的合成,PDGF在两种细胞中,也表现出有时程的差异。