Brain and spinal cord trauma:what we know about the therapeutic potential of insulin growth factor 1 gene therapy

Although little attention has been paid to cognitive and emotional dysfunctions observed in patients after spinal co rd injury,several reports have described impairments in cognitive abilities.Our group also has contributed significantly to the study of cognitive impairments in a rat model of spinal co rd injury.These findings are very significant because they demonstrate that cognitive and mood deficits are not induced by lifestyle changes,drugs of abuse,and combined medication.They are related to changes in brain structures involved in cognition and emotion,such as the hippocampus.Chronic spinal cord injury decreases neurogenesis,enhances glial reactivity leading to hippocampal neuroinflammation,and trigge rs cognitive deficits.These brain distal abnormalities are recently called te rtiary damage.Given that there is no treatment for Tertiary Damage,insulin growth factor 1 gene therapy emerges as a good candidate.Insulin growth factor 1 gene thera py recove rs neurogenesis and induces the polarization from pro-inflammato ry towards anti-inflammatory microglial phenotypes,which represents a potential strategy to treat the neuroinflammation that supports te rtiary damage.Insulin growth factor 1 gene therapy can be extended to other central nervous system pathologies such as traumatic brain injury where the neuroinflammatory component is crucial.Insulin growth factor 1 gene therapy could emerge as a new therapeutic strategy for treating traumatic brain injury and spinal cord injury.

Expression of insulin-like growth factor Ⅱ and its receptor in liver cells of chronic liver diseases

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Expression of insulin-like growth factor Ⅱ and its receptor in hepatocellular carcinogenesis

INTRODUCTIONInsulin-like growth factor Ⅱ(IGF-Ⅱ) is a mitogenic peptide of 74 kD and is mostly synthesized in fetal liver tissue .IGF-Ⅱ is believed to play an important role in fetal growth and development and is involved in cellular proliferation and differentiation[1-5]. Recently ,several researchers have reported increased expression of the IGF-Ⅱgene in human hepatocellular carcinoma (HCC) and adjacent non-cancerous liver tissues [6-10].

Reactivation of the insulin-like growth factor-Ⅱsignaling pathway in human hepatocellular carcinoma

Constitutive activation of the insulin-like growth factor (IGF)-signaling axis is frequently observed in human hepatocellular carcinoma(HCC).Especially the over- expression of the fetal growth factor IGF-Ⅱ,IGF-Ⅰ receptor(IGF-IR),and cytoplasmic downstream effectors such as insulin-receptor substrates(IRS)contribute to proliferation,anti-apoptosis,and invasive behavior. This review focuses on the relevant alterations in this signaling pathway and independent in vivo models that support the central role IGF-Ⅱsignaling during HCC development and progression.Since this pathway has become the center of interest as a target for potential anti-cancer therapy in many types of malignancies,various experimental strategies have been developed,including neutralizing antibodies and selective receptor kinase inhibitors,with respect to the specific and efficient reduction of oncogenic IGF-Ⅱ/IGF-IR-signaling.

Interplay between micro RNA-17-5p, insulin-like growth factor-Ⅱ through binding protein-3 in hepatocellular carcinoma

AIM: To investigate the effect of microR NA on insulinlike growth factor binding protein-3(IGFBP-3) and hence on insulin-like growth factor-Ⅱ(IGF-Ⅱ) bioavailability in hepatocellular carcinoma(HCC).METHODS: Bioinformatic analysis was performed using microrna.org, DIANA lab and Segal lab softwares. Total RNA was extracted from 23 HCC and 10 healthy liver tissues using mir Vana mi RNA Isolation Kit. microR NA-17-5p(miR-17-5p) expression was mimicked and antagonized in Hu H-7 cell lines using Hi Per Fect Transfection Reagent, then total RNA was extracted using Biozol reagent then reverse transcribed into cD NA followed by quantification of mi R-17-5p and IGFBP-3 expression using Taq Man real-time quantitative PCR. Luciferase reporter assay was performed to validate the binding of miR-17-5p to the 3'UTR of IGFBP-3. Free IGF-Ⅱ protein was measured in transfected Hu H-7 cells using IGF-Ⅱ ELISA kit. RESULTS: Bioinformatic analysis revealed IGFBP-3 as a potential target for miR-17-5p. Screening of miR-17-5p and IGFBP-3 revealed a moderate negative correlation in HCC patients, where mi R-17-5p was extensively underexpressed in HCC tissues(P = 0.0012), while IGFBP-3 showed significant upregulation in the same set of patients(P = 0.0041) compared to healthy donors. Forcing mi R-17-5p expression in Hu H-7 cell lines showed a significant downregulation of IGFBP-3 mR NA expression(P = 0.0267) and a significant increase in free IGF-Ⅱ protein(P = 0.0339) compared to mock untransfected cells using unpaired t-test. Luciferase assay validated IGFBP-3 as a direct target of mi R-17-5p; luciferase activity was inhibited by 27.5% in cells co-transfected with miR-17-5p mimics and the construct harboring the wild-type binding region 2 of IGFBP-3 compared to cells transfected with this construct alone(P = 0.0474).CONCLUSION: These data suggest that regulating IGF-Ⅱ bioavailability and hence HCC progression can be achieved through targeting IGFBP-3 via manipulating the expression of miR NAs.

Expression Analysis of the Insulin-Like Growth Factors Ⅰ and Ⅱ During Embryonic and Early Larval Development of Turbot(Scophthalmus maximus)

The insulin-like growth factors I and II(IGF-I and IGF-II) are important proteins involved in fish growth and development. Here, we report the isolation of IGF-II and expression analysis of IGFs in turbot Scophthalmus maximus, aiming to clarify their function in embryonic and larval development of fish. The deduced IGF-II gene is 808 bp in full length, which encodes a protein of 219 amino acids and is 93% similar with that of Paralichthys olicaceus in amino acid sequence. The tissue abundance and the expression pattern of IGFs in a turbot at early development stages were investigated via reverse transcription-polymer chain reaction. Result showed that the IGF-I and IGF-II genes were widely expressed in tissues of S. maximus. IGF-I was detected in all tissues except intestines with the highest level in liver, while IGF-II transcript presented in all tissues except muscle. At the stages of embryonic and larval development, the m RNA levels of IGFs sharply increased from the stage of unfertilized egg to post larva, followed by a decrease with larval development. However, there was an increase in IGF-I at the embryonic stage and IGF-II at the gastrula stage, respectively. These results suggested that IGFs play important roles in cell growth and division of the turbot. Our study provides reference data for further investigation of growth regulation in turbot, which can guarantee better understanding of the physiological role that IGFs play in fish.

The immunocytochemical localization of insulin-like growth factor Ⅱ in human cirrhosis

Sections of 30 cases of human cirrhosis were stained with rabbit anti-insulin-likegrowth factor Ⅱ(IGF Ⅱ)by double PAP method.By the serological examination 15 patientsshowed HBV infection and sections of 14 eases were HBsAg postively with a total rate of 67％(20 cases).The IGF Ⅱ was positive in the cytoplasm of all the liver and ductular cells.Binucle-ated,polypoid liver cells and the peripheral cells of the lobules or nodules were distinctly posi-tive,The liver cells which were strongly positive were a kind of thin polygonal cells with asmall oval or a round deeply stained nucleus in each.They might exist sporadically in the lob-ules or in the marginal portion of a nodule.These liver cells are quite different from the so-called oval cells which are derived from the proliferating ductules and are generally believed tobe responsible for the pathogensis of hepatoma.

Antisense oligonucleotide to insulin-like growth factorⅡ induces apoptosis in human ovarian cancer AO cellline

The effects of antisense oligonucleotide to insulin-like growth factor 11 (IGFII) to induce apoptosis in human ovarian cancer cells were evaluated. Antiproliferation effects of antisense to IGFII in ovarian cancer AO cells were determined by 3H-thymidine incorporation. Apoptosis of the IGFll antisense-treated cells was quantitated by both nuclear condensation and flow cytometry after cells were stained with propidium iodide. IGFII antisense (4.5μM)treatment of 48 h maximally inhibited proliferation of AO cells. More than 25% of IGFII antisense-treated cells (4.5PM for 24 h) had undergone apoptosis, whereas less than 3% of the cells were apoptotic in either IGFII sense-treatedcells or untreated cells. Antisense oligonucleotide to IGFII significantly inhibited cell proliferation and induced apoptosis in human ovarian cancer AO cell. These data suggest that IGFII may be a potential target in treatment of ovarian cancer and antisense oligonucleotide to IGFⅡmay serve as a therapeutic approach.

Changes of insulin-like growth factor-Ⅱ and insulin-like growth factor binding protein-3 in cerebrospinal fluid of children with tuberculous meningitis

BACKGROUND： Recent studies have found that insulin-like growth factors （IGFs） and insulin-like growth factor binding protein-3 （IGFBP-3） have stronger neurotrophic and neuroprotective effects. But whether their levels in cerebrospinal fluid could be used as an auxiliary indicator in differentially diagnosing tuberculous meningitis and viral encephalitis is not yet clear. OBJECTIVE： To explore the changes of insulin-like growth factor-Ⅱ （IGF-Ⅱ ） and IGFBP-3 in cerebrospinal fluid （CSF） of children with tuberculous meningitis and the significance of the changes. DESIGN： A non-randomized concurrent controlled study. SETTING： Department of Pediatric Internal Medicine, the First Affiliated Hospital of Xinxiang Medical College. PARTICIPANTS： Thirty children with tuberculous meningitis （14 males and 16 females） were selected from the Department of Pediatric Internal Medicine, the First Affiliated Hospital of Xinxiang Medical College from January 2005 to December 2006. Tuberculous meningitis was diagnosed according to their clinical manifestations, the history of close contact with tuberculosis, typical cerebrospinal fluid changes of tuberculous meningitis, positive tuberculosis antibody and effective antituberculosis treatment. There were 30 children （13 males and 17 females） with viral encephalitis, and viral encephalitis was diagnosed according to epidemiological history, clinical manifestations, conventional and biochemical changes of cerebrospinal fluid, and negative bacteriology judgment. Meanwhile, 30 children （13 males and 17 females） without infectious and central nervous system disease were selected as the control group. Informed consent was obtained from the parents of all the enrolled children. METHODS： ①The lumbar puncture operation was implemented immediately to obtain cerebrospinal fluid （3 mL）. The contents of IGF-Ⅱ and IGFBP-3 were detected with immunoradiometric assay. The concentrations of glucose and protein in cerebrospinal fluid were determined with a dry-chemical method. The number of white blood cells was counted by Fushi Method. ②The Pearson correlation analysis was used to analyze the correlation of the contents of IGF-Ⅱ and IGFBP-3 in cerebrospinal fluid with the leucocyte counting and the concentrations of glucose and protein in cerebrospinal fluid. MAIN OUTCOME MEASURES： The contents of IGF- Ⅱ and IGFBP-3 in cerebrospinal fluid, and their correlation with the leucocyte counting and the concentrations of glucose and protein in cerebrospinal fluid. RESULTS： ①Contents of IGF-Ⅱ and IGFBP-3 in cerebrospinal fluid： The contents of IGF-Ⅱ and IGFBP-3 in cerebrospinal fluid in the tuberculous meningitis group were significantly higher than those in the encephalitis virus group and control group （P 〈 0.05）. There was no significant difference in the contents of IGF- Ⅱ and IGFBP-3 in cerebrospinal fluid between the viral encephalitis group and control group （P 〉 0.05）. ②Correlation： The IGF- Ⅱ and IGFBP-3 contents in cerebrospinal fluid were positively correlated with the protein concentration in cerebrospinal fluid （r =0.821, 0.855, P 〈 0.01）, but negatively with the glucose （r =0.742, - 0.605, P 〈 0.01）. CONCLUSION- ①IGFs and IGVBPs are involved in the pathophysiological process of tuberculous meningitis, as well as the glucose and protein metabolism in cerebrospinal fluid. ②The IGF-Ⅱ and IGFBP-3 contents in cerebrospinal fluid can be used as the auxiliary indicators to differentially diagnose tuberculous meningitis and viral enceohalitis.

EXPRESSION OF INSULIN-LIKE GROWTH FACTOR Ⅱ(IGF-Ⅱ)IN HUMAN HEPATOCELLULAR CARCINOMA AND LIVER CIRRHOSIS:ITS RELATIONSHIP WITH HEPATITIS B VIRUS X PROTEIN EXPRESSION

Sixty cases of hepatocellular carcinoma (HCC) and 47 cases of liver cirrhosis (LC) were examined with immunocytochemistry method using antibodies against IGF-II and HBxAg on formalin-fixed, paraffin-embedded tissue sections. 32 HCC and 37 LC were found to be positive to HBxAg, in which the positive rates of IGF-II were 100% (32/32) and 94.6% (35/37) respectively. 28 HCC and 10 LC were found to be HBxAg negative, IGF-II was positive in 23 HCC (83.1%) and 6 LC (60%). The positive expression rates of IGF-II in HBxAg positive tissues were significantly higher than those in HBxAg negative tissues (P<0.05). There were three types of distribution of IGF-II expression in HCC and LC: (1) perinucleus; (2) diffuse in cytoplasm; (3) inside nucleus. IGF-II was highly expressed in most of hyperplastic and neoplastic nodules hepatocytes and some of regeneration nodules. Small polygonal liver cells (SPLCs) were found in the liver tissues surrounding the tumor and cirrhosis and they were positive to both IGF-II and HBxAg. The positive rates of IGF-II in SPLC were 86.4% (38/44) in the HBxAg-positive tissues and 40.5%, (15/37) in the HBxAg-negative tissues. The above findings suggest that IGF-II plays an important role in abnormal proliferation of HCC and SPLC. The relation between IGF-II andHBxAg and the nature of SPLCs are also discussed.

达格列净抑制血管紧张素Ⅱ诱导的心肌细胞肥大和凋亡

目的探讨达格列净对血管紧张素Ⅱ(AngⅡ)诱导的心肌细胞肥大反应和凋亡的影响。方法分离培养原代大鼠乳鼠心肌细胞,将其随机分为4组:对照组,AngⅡ组、达格列净1组(0.5μmol/L),达格列净2组(2μmol/L)。采用α-actin染色检测细胞面积,采用qPCR检测胚胎基因的转录,采用Tunel染色检测细胞凋亡水平,采用caspase3试剂盒检测caspase3活性,采用免疫印迹检测经典信号分子。结果AngⅡ组细胞面积明显大于对照组(P<0.05);达格列净1组、达格列净2组细胞面积低于AngⅡ组(P<0.05)。qPCR结果显示AngⅡ组胚胎基因转录明显高于对照组(P<0.05);达格列净1组,达格列净2组胚胎基因转录低于AngⅡ组(P<0.05)。tunel染色结果显示:AngⅡ组细胞凋亡数量高于对照组(P<0.05);达格列净1组,达格列净2组细胞凋亡数量低于AngⅡ组(P<0.05)。AngⅡ组细胞caspase3活性高于对照组(P<0.05);达格列净1组,达格列净2组细胞caspase3活性低于AngⅡ组(P<0.05)。免疫印迹检测结果显示AngⅡ组细胞胰岛素样生长因子1受体(IGF1R)和Akt激活程度低于对照组(P<0.05);达格列净1组,达格列净2组细胞IGF1R和Akt激活高于AngⅡ组(P<0.05)。结论达格列净可直接作用于心肌细胞,保护其免受AngⅡ诱导的损伤。

母乳IGF-Ⅱ与婴儿体格生长指标关联性分析及影响因素研究

目的描述哺乳期各阶段母乳IGF-Ⅱ浓度的变化,探讨哺乳期不同阶段母乳IGF-Ⅱ与婴儿体格生长指标的关联,以及可能影响母乳IGF-Ⅱ浓度的因素。方法2020年10月至2021年9月于北京大学人民医院招募32名满足纳入排除标准的分娩妇女,分别于分娩后48 h、产后15 h、产后42 h、6个月、9个月及12个月采集妇女母乳标本,同时收集婴儿身长、体重等生长发育指标。采用酶联免疫吸附方法检测母乳IGF-Ⅱ水平。使用广义估计方程(GEE)分析母乳IGF-Ⅱ水平与婴儿体格生长指标之间的关联以及可能影响母乳IGF-Ⅱ水平的因素。结果母乳IGF-Ⅱ中位浓度在12月成熟乳中最高(82.5 ng/mL),6月成熟乳中最低(55.3 ng/mL),呈先下降再升高趋势,但各阶段中位浓度间未见明显差异;在调整母亲年龄、孕前BMI、孕周、母亲民族、孕产史、孕期并发症情况(妊娠期糖尿病、妊娠期高血压、妊娠期甲状腺疾病)、婴儿喂养方式及婴儿性别后,研究结果显示初乳中IGF-Ⅱ含量与婴儿身长呈正相关关系(β=0.9,P<0.01),初乳、42d成熟乳、12月成熟乳中的IGF-Ⅱ含量与婴儿体重呈正相关关系(P=0.02,P<0.01和P=0.01)。将膳食炎症指数(DII)按三分位法分为三组(Q1:最抗炎倾向组,Q2:中间组,Q3:最促炎倾向组)纳入GEE模型,结果显示,第三分位组(即最促炎倾向组)与母乳IGF-Ⅱ含量正相关(β=28.6,P=0.01)。结论母乳IGF-Ⅱ对婴儿身长、体重存在一定的促进作用,孕期促炎饮食与母乳IGF-Ⅱ含量相关。

Targeting the insulin-like growth factor pathway in hepatocellular carcinoma

Hepatocellular carcinoma(HCC) is the third leading cause of cancer-related deaths worldwide. Only 30%-40% of the patients with HCC are eligible for curative treatments, which include surgical resection as the first option, liver transplantation and percutaneous ablation. Unfortunately, there is a high frequency of tumor recurrence after surgical resection and most HCC seem resistant to conventional chemotherapy and radiotherapy. Sorafenib, a multi-tyrosine kinase inhibitor, is the only chemotherapeutic option for patients with advanced hepatocellular carcinoma. Patients treated with Sorafenib have a significant increase in overall survival of about three months. Therefore, there is an urgent need to develop alternative treatments. Due to its role in cell growth and development, the insulin-like growth factor system is commonly deregulated in many cancers. Indeed, the insulin-like growth factor(IGF) axis has recently emerged as a potential target for hepatocellular carcinoma treatment. To this aim, several inhibitors of the pathway have been developed suchas monoclonal antibodies, small molecules, antisense oligonucleotides or small interfering RNAs. However recent studies suggest that, unlike most tumors, HCC development requires increased signaling through insulin growth factor Ⅱ rather than insulin growth factor Ⅰ. This may have great implications in the future treatment of HCC. This review summarizes the role of the IGF axis in liver carcinogenesis and the current status of the strategies designed to target the IGF-Ⅰ signaling pathway for hepatocellular carcinoma treatment.

Influence of Intensive Insulin Therapy on Vascular Endothelial Growth Factor in Patients with Severe Trauma

The influence of early-stage intensive insulin therapy on the plasma levels of vascular en- dothelial growth factor （VEGF） and the related parameters in patients with severe trauma and the clini- cal implication were investigated. Sixty-four cases of severe trauma （injury severity score 〉20） with stress hyperglycemia （blood glucose 〉9 mmol/L） were randomly divided into intensive insulin therapy group and conventional therapy group. ELISA method, radioimmunoassay and density gradient grada- tion one-step process were used to determine plasma VEGF, endothelin-1 （ET-1）, and the number of circulating endothelial cells （CECs） at the day of 0, 2, 3, 5 and 7 after admission. Simultaneously, the changes of CRP concentration in plasma were monitored to evaluate inflammatory response. The results showed that plasma levels of observational indexes in patients receiving early-stage intensive insulin therapy were all significantly lower than those in conventional therapy groups 2, 3, 5 and 7 days after admission [for VEGF （ng/L）, 122.2±23.8 vs. 135.9±26.5, 109.6±27.3 vs. 129.0±18.4, 88.7±18.2 vs. 102.6±27.3, 54.2±26.4 vs. 85.7±35.2, P〈0.05, 0.01, 0.05, 0.05 respectively; for ET-1 （ng/L）, 162.8±23.5 vs. 173.7±13.2, 128.6±17.5 vs. 148.8±22.4, 96.5±14.8 vs. 125.7±14.8, 90.7±16.9 vs. 104.9±22.5, P〈0.05, 0.01, 0.01, 0.01 respectively; for CRP （mg/L）, 23.2±13.8 vs. 31.9±16.5, 13.6±17.3 vs. 23.5±18.4, 8.7±10.2 vs. 15.6±13.3, 5.2±9.4 vs. 10.7±11.2, all P〈0.05; for CECs （/0.9 μL）, 10.9±5.6 vs. 13.9±6.2, 8.5±4.9 vs. 11.3±5.3, 6.3±6.4 vs. 9.4±5.7, 4.8±7.1 vs. 7.8±4.8, all P〈0.05]. It was concluded that intensive insulin therapy could antagonize the endothelium injury after trauma and reduce inflammation response quickly, which was one of important mechanisms by which intensive insulin therapy improves the prognosis of trauma patients.

Insulin-like growth factor-1 induces lymphangiogenesis and facilitates lymphatic metastasis in colorectal cancer

AIM:To investigate the expression of insulin-like growth factor-1(IGF-1)/insulin-like growth factor-1 receptor(IGF-1R)in colorectal cancer(CRC)tissues and to analyze their correlation with lymphangiogenesis and lymphatic metastasis.METHODS:Immunohistochemistry was used to evaluate IGF-1 and IGF-1R expression and lymphatic vessel density(LVD)in 40 CRC specimens.The correlation between IGF-1/IGF-1R and LVD was investigated.Effects of IGF-1 on migration and invasion of CRC cells were examined using transwell chamber assays.A LoVo cell xenograft model was established to further detect the role of IGF-1 in CRC lymphangiogenesis in vivo. RESULTS:Elevated IGF-1 and IGF-1R expression in CRC tissues was correlated with lymph node metastasis(r=0.715 and 0.569,respectively,P<0.05)and tumor TNM stage(r=0.731 and 0.609,P<0.05).A higher LVD was also found in CRC tissues and was correlated with lymphatic metastasis(r=0.405,P<0.05).A positive correlation was found between LVD and IGF-1R expression(r=0.437,P<0.05).Transwell assays revealed that IGF-1 increased the migration and invasion of CRC cells.In vivo mouse studies showed that IGF-1 also increased LVD in LoVo cell xenografts.CONCLUSION:IGF-1/IGF-1R signaling induces tumorassociated lymphangiogenesis and contributes to lymphatic metastasis of CRC.

Integrating insulin-like growth factor 1 and sex hormones into neuroprotection:Implications for diabetes

Brain integrity and cognitive aptitude are often impaired in patients with diabetes mellitus, presumably a result of the metabolic complications inherent to the disease. However, an increasing body of evidence has demonstrated the central role of insulin-like growth factor 1(IGF1) and its relation to sex hormones in many neuroprotective processes. Both male and female patients with diabetes display abnormal IGF1 and sexhormone levels but the comparison of these fluctuations is seldom a topic of interest. It is interesting to note that both IGF1 and sex hormones have the ability to regulate phosphoinositide 3-kinase-Akt and mitogen-activated protein kinases-extracellular signal-related kinasesignaling cascades in animal and cell culture models of neuroprotection. Additionally, there is considerable evidence demonstrating the neuroprotective coupling of IGF1 and estrogen. Androgens have also been implicated in many neuroprotective processes that operate on similar signaling cascades as the estrogen-IGF1 relation. Yet, androgens have not been directly linked to the brain IGF1 system and neuroprotection. Despite the sex-specific variations in brain integrity and hormone levels observed in diabetic patients, the IGF1-sex hormone relation in neuroprotection has yet to be fully substantiated in experimental models of diabetes. Taken together, there is a clear need for the comprehensive analysis of sex differences on brain integrity of diabetic patients and the relationship between IGF1 and sex hormones that may influence brain-health outcomes. As such, this review will briefly outline the basic relation of diabetes and IGF1 and its role in neuroprotection. We will also consider the findings on sex hormones and diabetes as a basis for separately analyzing males and females to identify possible hormone-induced brain abnormalities. Finally, we will introduce the neuroprotective interplay of IGF1 and estrogen and how androgen-derived neuroprotection operates through similar signaling cascades. Future research on both neuroprotection and diabetes should include androgens into the interplay of IGF1 and sex hormones.

Improvement in erectile dysfunction after insulin-like growth factor-1 gene therapy in diabetic rats

Aim： To determine whether adenoviral gene transfer of insulin like growth factor-1 （IGF-1） to the penis of streptozotocin （STZ）-induced diabetic rats could improve erectile capacity. Methods： The STZ diabetic rats were transfected with AdCMV-βgal or AdCMV-IGF-1. These rats underwent cavernous nerve stimulation to assess erectile function and their responses were compared with those of age-matched control rats 1 to 2 days after transfection. In control and transfected STZ diabetic rats, IGF-1 expression were examined by reverse transcription polymerase chain reaction （RT-PCR）, Western blot and histology. The penis β-galactosidase activity and localization of the STZ diabetic rats were also determined. Results： One to two days after transfection, the β-galactosidase was found in the smooth muscle cells of the diabetic rat penis transfected with AdCMV-βgal. One to 2 days after administration of AdCMV- IGF-1, the cavernosal pressure, as determined by the ratio of maximal intracavernous pressure-to-mean arterial pressure （ICP/MAP） and total intracavernous pressure （ICP）, was increased in response to cavernous nerve stimulation. Transgene expression was confirmed by RT-PCR, Western blot and histology. Conclusion： Gene transfer of IGF-1 significantly increased erectile function in the STZ diabetic rats. These results suggest that in vivo gene transfer of IGF- 1 might be a new therapeutic intervention for the treatment of erectile dysfunction （ED） in the STZ diabetic rats.

Effect of endogenous insulin-like growth factor and stem cell factor on diabetic colonic dysmotility

AIM: To investigate whether the reduction of stem cell factor (SCF) is mediated by decreased endogenous insulin-like growth factor (IGF)-1 in diabetic rat colon smooth muscle. METHODS: Sixteen Sprague-Dawley rats were randomly divided into two groups: control group and streptozotocin-induced diabetic group. After 8 wk of streptozotocin administration, colonic motility function and contractility of circular muscle strips were measured. The expression of endogenous IGF-1 and SCF was tested in colonic tissues. Colonic smooth muscle cells were cultured from normal adult rats. IGF-1 siRNA transfection was used to investigate whether SCF expression was affected by endogenous IGF-1 expression in smooth muscle cells, and IGF-1 induced SCF expression effects were studied. The effect of high glucose on the expression of endogenous IGF-1 and SCF was also investigated. RESULTS: Diabetic rats showed prolonged colonic transit time (252 ± 16 min vs 168 ± 9 min, P < 0.01) and weakness of circular muscle contraction (0.81 ± 0.09 g vs 2.48 ± 0.23 g, P < 0.01) compared with the control group. Endogenous IGF-1 and SCF protein expression was significantly reduced in the diabetic colonic muscle tissues. IGF-1 and SCF mRNA expression also showed a paralleled reduction in diabetic rats. In the IGF-1 siRNA transfected smooth muscle cells, SCF mRNA and protein expression was significantly decreased. IGF-1 could induce SCF expression in a concentration and time-dependent manner, mainly through the extracellular-signal-regulated kinase 1/2 signal pathway. High glucose inhibited endogenous IGF-1 and SCF expression and the addition of IGF-1 to the medium reversed the SCF expression. CONCLUSION: Myopathy may resolve in colonic motility dysfunction in diabetic rats. Deficiency of endogenous IGF-1 in colonic smooth muscle cells leads to reduction of SCF expression.

Effects of Tanshinone ⅡA on Transforming Growth Factor β1-Smads Signal Pathway in Renal Interstitial Fibroblasts of Rats

The effects of tanshinone ⅡA （TSN） on transforming growth factor β1 （TGFβ1） signal transduction in renal interstitial fibroblasts of rats were studied in order to investigate its mechanism in prevention of renal interstitial fibrosis. Rat renal fibroblasts of the line NRK/49F were cultured in vitro, stimulated with 5 ng/mL TGFβ1 and pretreated with 10-6, 10-5, 10-4 mol/L TSN respectively. The mRNA levels of fibronectin （FN） were examined by RT-PCR. The protein expression of FN and Smads was detected by Western blot. TGFβ1 induced the expression of FN mRNA and Smads in a time-dependent manner in a certain range. Compared with pre-stimulation, the FN mRNA and protein levels were increased by 1.1 times and 1.5 times respectively （P〈0.01, P〈0.01）, and the protein expression of phosphorylated Smad2/3 （p-Smad2/3） increased by 7 times at the end of TGFβ1 stimulation （P〈0.01）. TSN pretreatment may down-regulate the FN and p-Smad2/3 expression in a dose-dependent manner. 10-6 mol/L TSN pretreatment had no effect on the FN and p-Smad2/3 expression （both P〉0.05）. After pretreatment with 10-5 and 10-4 mol/L TSN, the FN mRNA levels were decreased by 28.1% and 43.8% respectively （P〈0.05, P〈0.01）, the FN protein levels were decreased by 40% and 44% respectively （P〈0.05, P〈0.05）, and the p-Smad2/3 protein expression were decreased by 40% and 65% respectively （P〈0.05, P〈0.01）. The inhibitory effect of TSN on renal interstitial fibrosis may be related to its blocking effect on TGFβ1-Smads signal pathway in renal intersti- tial fibroblasts.

Insulin-like Growth Factor-Ⅰ Gene Cloning and Protein Expression in Bovine Trabecular Meshwork Tissue and Cells

Whether cultured bovine trabecular meshwork cells and trabecular tissue ex vivo express insulin like growth factor I (IGF Ⅰ) messenger RNA (mRNA) and protein was investigated. Total RNA of cultured bovine trabecular meshwork cells as well as trabecular meshwork tissue freshly excised from bovine eyes was extracted, and reverse transcriptase polymerase chain reaction (RT PCR) was used to detect IGF Ⅰ mRNA. RT PCR product was verified by sequencing. Immunohistochemical stain was used to detect IGF Ⅰ protein. The results showed that a single PCR amplified product was obtained, and the sequence was homologous to the known sequence.. IGF Ⅰ immunostain was positive in the cytoplasm of trabecular meshwork cells. It was concluded that trabecular meshwork cells produce IGF Ⅰ and contribute to the presence of IGF Ⅰ in trabecular meshwork microenvironment as well as aqueous humor. Trabecular meshwork cells were affected by IGF Ⅰ not only through paracrine, but also autocrine action. Whether abnormal down regulations in IGF Ⅰ production may contribute to the pathogenesis of primary open angle glaucoma and the possibility of promoting the autocrine action of IGF Ⅰ by trabecular meshwork cells to treat the diesease is worth further investigation.