体外垂体生长激素腺瘤转录靶向性基因治疗的实验研究

目的评价人生长激素启动子调控的基因治疗系统对垂体生长激素腺瘤的靶向性治疗作用。方法构建生长激素启动子调控的基因系统，体外转染垂体生长激素腺瘤GH3细胞；观察治疗基因HSV—TK在细胞中的表达靶向性，以及该系统杀伤细胞的能力和杀伤靶向性。结果HSV—TK蛋白能够在GH3细胞中靶向性表达，予以更昔洛韦后，GH3细胞增殖速度明显减慢，而对照细胞无明显杀伤作用（P〈0．01）。结论生长激素启动子调控的基因治疗系统能有效地、靶向性地抑制GH3细胞增殖。     

In vivo insulin sensitivity in adrenodemedullated rats

Insulin sensitivity and responsiveness were determined in adrenal demedullated rats (ADMX) with euglycemic insulin clamp technique. Adrenal medulla was extirpated bilaterally a week before the study. Catheters were placed at right atrium via right jugular vein for sampling blood and at inferior vena cava via femoral vein for the infusion of insulin and glucose solution. Insulin was infused at rates of 4.4, 8.8, 14.7, 29.3, 88.0, 293.0 mU/kg/min. Blood was collected every five min. during the clamp and glucose infusion rate was modulated to control the blood glucose concentrations at fasting levels. Glucose metabolism was calculated from the amount of glucose infused from 60th to 120th min. during the euglycemic clamp. The results obtained were as follows: 1. Glucose metabolisms of ADMX in each infusion rate of insulin, 4.4, 8.8, 14.7, 29.3, 88.0, 293.0 mU/kg/min were 5.2 +/- 0.5, 12.5 +/- 0.5, 17.6 +/- 1.2, 19.8 +/- 2.3, 29.0 +/- 1.5, and 25.2 +/- 1.9 mg/kg/min, respectively. 2. Glucose metabolisms of control group in each dose were 6.6 +/- 0.4, 9.0 +/- 0.9, 18.5 +/- 1.2, 23.4 +/- 2.4, 24.6 +/- 1.1, and 27.0 +/- 1.3 mg/kg/min, respectively. 3. Significant difference (p less than 0.01) in glucose metabolism between ADMX and control was observed at the insulin infusion rate of 8.8 mU/kg/min which might be equivalent to physiological hyperinsulinemia. 4. There were not any differences in insulin responsiveness between both groups. These results suggest that epinephrine regulates insulin sensitivity under physiological hyperinsulinemic condition via defects of insulin receptors.     

In vitro effect of insulin on rats erythrocytes rheological behaviour

Blood viscosity is an important cardiovascular risk factor that might be related to diabetes complications. Hyperinsulinemia has been suggested as "the most important candidate" to characterise diabetes as a risk factor for cardiovascular disease. There is no evidence of the beneficial effect of insulin on type 2 diabetes erythrocytes in patients without cardiovascular disease, whereas the opposite is observed in those with cardiovascular disease. In the present study we analysed the in vitro effect of different doses of insulin on red blood cell rheological aspects in an obesity model. Previous studies carried out in beta strain rats had shown that this strain possess insulin blood levels higher than the ones observed in alpha strain (eumetabolic), as well as blood hyperviscosity and erythrocyte deformability decrease. Our results points out that in vitro insulin produced an increase in erythrocyte aggregability, although it did not modified either their osmotic fragility or erythrocyte deformability estimated by viscometry, even against decreased viscosity of treated erythrocytes submitted to increased shear rate.     

Increase in T-type calcium current in atrial myocytes from adult rats with growth hormone-secreting tumors.

Growth hormone (GH) has pronounced effects on protein synthesis and cell growth in cardiac muscle from adult animals, although the mechanism of its action is not understood. Because Ca2+ has been implicated as a regulator of mitogenic processes in a number of tissues, we investigated whether GH affects the transmembrane movement of Ca2+ through voltage-activated channels of cardiac myocytes. Atrial and ventricular myocytes were isolated from adult rats with GH-secreting tumors and studied electrophysiologically by using patch-clamp techniques. Tumor-bearing rats re-enter an active growth phase and double their body weight over age-matched controls 8 weeks after introduction of the tumor. Atrial myocytes from tumor-bearing animals showed a 3-fold increase in the density of T-type Ca2+ current compared with cells from control animals, although the voltage dependency of activation and inactivation of T-type current was not altered. The increase in T-current density of atrial myocytes preceded by at least a week any measurable change in heart weight, body weight, or myocyte size. L-type Ca2+ currents in atrial and ventricular cells were not affected. The results suggest that a tumor-derived growth factor, most likely GH, can cause a specific enhancement of T-type Ca2+ current in atrial myocytes.     

Microalbuminuria in insulin sensitivity in patients with growth hormone-secreting pituitary tumor

CONTEXT: Impaired glucose tolerance and diabetes mellitus are frequently present in acromegalic patients in whom the degree of impaired glucose metabolism seems directly correlated with GH levels. Microalbuminuria is reported to be directly correlated with insulin resistance, and both conditions predict cardiovascular disease mortality. OBJECTIVE: Our objective was to investigate the microalbuminuria levels as a marker of endothelial dysfunction in acromegalic patients. DESIGN: We conducted an observational, multicenter, open prospective study. SUBJECTS: Subjects included 74 patients with active acromegaly (52 with normal glucose tolerance, 16 with impaired glucose tolerance, and six with diabetes), and 50 healthy subjects matched for age, gender, and body mass index were studied as controls. RESULTS: In the whole group, mean GH and IGF-I levels were 24.2+/-3.9 ng/ml and 700.1+/-23.0 microg/liter, respectively. The insulin sensitivity index (ISI) in the patients was lower than in the controls (P<0.0005). In impaired glucose tolerance and diabetic patients, microalbuminuria was higher than in normal glucose tolerance patients (P<0.05 and P<0.0005 respectively). Hypertensive patients had higher levels of microalbuminuria than normotensive ones (P<0.005). The levels of microalbuminuria related to creatinine were directly correlated with fasting glucose levels (r=0.27; P=0.0019), fasting insulin levels (r=0.28; P=0.017), and insulin after 90 (r=0.26; P=0.027) and 120 min after glucose load (r=0.26; P=0.023) and indirectly correlated with ISI composite (P<0.0001; r=-0.48). By a multivariate analysis, the log-ISI composite was the strongest predictor of microalbuminuria (t=-3.19; P=0.0021). CONCLUSIONS: Impairment of glucose tolerance in acromegaly is associated with high levels of microalbuminuria. For this reason, microalbuminuria should be part of cardiovascular risk assessment in these patients.     

In vitro effect of human recombinant leptin and expression of leptin receptors on growth hormone-secreting human pituitary adenomas

OBJECTIVES: Untreated growth hormone deficiency (GHD) is implicated in the increased cardiovascular risk associated with adult hypopituitarism. Oxidative stress, predisposing to lipid peroxidation, may be an important mediator of endothelial dysfunction, a pro-atherogenic state associated with adult GHD. DESIGN AND PATIENTS: In a randomized, double-blind, placebo-controlled study we investigated the effects of GH replacement on low-density lipoprotein (LDL) oxidation and neutrophil superoxide (O(-)(2)) generating capacity in 32 GHD adults (19 males, 13 females; age range 19-64 years) over 3 months. Thirty age- and sex-matched healthy controls were also studied. MEASUREMENTS: Lipid hydroperoxides (HPOs) in plasma were measured using the ferrous oxidation with xylenol orange (FOX) assay. The susceptibility of LDL to oxidation was assessed by the copper-catalysed lag phase of LDL oxidation. Neutrophil O(-)(2)- generating capacity was assessed by a lucigenin-based chemiluminescent assay of NADPH oxidase activity. Body composition was assessed using bioelectrical impedance analysis. RESULTS: Compared to controls, GHD subjects had higher LDL cholesterol (4.0 +/- 0.8 vs. 3.5 +/- 0.9 mmol/l, P < 0.01) and higher triglyceride concentrations (2.3 +/- 1.5 vs. 1.1 +/- 0.7 mmol/l, P < 0.001) but lower HDL cholesterol (1.1 +/- 0.3 mmol/l vs. 1.4 +/- 0.4 mmol/l, P < 0.01), lower levels of HPOs (0.72 +/- 0.35 vs. 0.92 +/- 0.20 microm, P < 0.01) and lower basal (2.5 +/- 1.5 vs. 4.5 +/- 2.3 mV/5 x 10(5) neutrophils, P < 0.01) and peak post-activation levels (23.2 +/- 11.1 vs. 34.4 +/- 15.6 mV/5 x 10(5) neutrophils, P < 0.01) of neutrophil O(-)(2)- generation. GH replacement resulted in an increase in HPOs from 0.70 +/- 0.39 to 0.86 +/- 0.19 microm (P < 0.05), although there was no change in the lag time of LDL oxidation. Neutrophil O(-)(2)- generating capacity was enhanced with a rise in basal O(-)(2)- generation from 2.8 +/- 1.4 to 5.4 +/- 4.6 mV/5 x 10(5) neutrophils (P < 0.05) and in peak post-activation O(-)(2)- generation from 21.9 +/- 9.5 to 35.8 +/- 21.7 mV/5 x 10(5) neutrophils (P < 0.05). LDL cholesterol was reduced from 4.1 +/- 0.8 mmol/l to 3.5 +/- 0.8 mmol/l (P < 0.01). No significant changes in measured parameters occurred in the placebo group. CONCLUSIONS: Adult GHD is associated with reduced lipid peroxidation and impaired neutrophil O(-)(2)- generating capacity, both of which are reversible with GH replacement. Our data suggest that: (i) that oxidative stress is not a major feature of the pro-atherogenic state in hypopituitary adults with GHD and (ii) a role for GH in modulating neutrophil function and leucocyte-lipoprotein interactions.     

In vivo and in vitro suppression by leptin of glucose-stimulated insulin hypersecretion in high glucose-fed rats

OBJECTIVES: Chronic feeding to rats of high glycaemic index (GI) diets results in the hypersecretion of insulin in response to an i.v. glucose load. The first aim of this study was to see if this exaggerated insulin response was accompanied by a hypersensitivity to glucose stimulation in isolated islets in vitro. The second aim was to see if the adipocyte factor, leptin, was able to alter insulin secretion in this model both in vivo and in vitro. DESIGN AND METHODS: Rats were fed for 6 weeks either a high GI diet in which the carbohydrate component was mostly glucose (GLUC diet) or a low GI diet containing mostly amylose (AMOSE diet). Rats then underwent an i.v. glucose tolerance test (ivGTT) (1g/kg) with and without a prior infusion of leptin (133 microg/kg perh). Islets were then isolated from these rats and basal and glucose-stimulated insulin secretion (GSIS) measured in both the absence and presence (100ng/ml) of leptin. RESULTS AND CONCLUSIONS: Peak insulin response during the ivGTT was 3-fold greater in GLUC rats (P<0.001). Leptin had no effect on AMOSE rat insulin response but lowered the GLUC rat response to AMOSE rat levels. In vitro, basal insulin secretion was 4-fold greater in GLUC rats (P<0.05). At 20mmol/l glucose, there was no further increase in insulin secretion in GLUC rats but a 2-fold increase in AMOSE rats. Leptin had no effect on basal insulin secretion or GSIS in AMOSE rats but reduced basal insulin secretion and GSIS in GLUC rats. These results show insulin hypersecretion in high GI-fed rats may be reduced by leptin.     

Cardiomegaly and haemodynamics in rats with a transplantable growth hormone-secreting tumour

To investigate cardiovascular changes in experimental acromegaly, a growth hormone-secreting tumour (MtT-W-15) was implanted in adult female rats. Somatic and tumour growth occurred steadily during the 8 week study period, as did an increase in serum growth hormone titre. Weight of left ventricle and right ventricle increased directly with tumour growth, both on an absolute basis and when compared with normal rats of equal body weight. Atrial weight also increased substantially. Haematocrit declined sharply at first, and more slowly later with increasing tumour weight. Haemodynamic measurements were made on these animals at two stages of tumour growth using an anaesthetised open-chest preparation. Cardiac index (per g body wt), stroke index, stroke work, left ventricle +dP/dtmax, and dF/dtmax of aortic flow were greatly elevated in rats with the largest tumours (longer duration), and to a lesser extent in those with smaller tumours (shorter duration). Systemic peripheral resistance and heart rate were depressed. Ventricular weight increased non-linearly with increases in cardiac index. Cardiac output, stroke volume, stroke work and dF/dtmax normalised per g left ventricle weight were also elevated. Splenomegaly accompanied tumour growth; however, splenectomy of tumour-bearing animals failed to prevent development of anaemia and cardiomegaly. While a direct effect of elevated growth hormone provides the best explanation for development of cardiomegaly in this model, volume work overloading due to anaemia and water retention may be a contributory cause.     

In vivo induction of neoplastic growth in nude mouse connective tissue adjacent to xenografted human tumors

Summary Induction of neoplastic growth of murine stroma cells within the human tumor xenograft was observed after serial passage of CEA and 2-microglobulin producing human colonic SLu tumor xenografts in nu/nu BALB/c mice. Mouse tumors within the human tumor xenografts wre identified using specific immunohistologic staining techniques for mouse histocompatibility marker or human CEA. These mixed tumors could be distinguished from normal human tumor xenografts by a different relationship between development of the tumor marker in the serum and tumor size. We were able to establish transformed murine cells from human xenografts, either induced by SC injection of 1×10⁶ tumor cells of the SLu cell line or by human SLu or mammary carcinoma tissue serially passaged in athymic animals. The established human and murine cell lines were characterized by cytogenetic methods. Transformed murine cells were then continuously passaged in tissue culture. The transformed mouse fibroblasts proved to possess tumorigenicity in nude mice. In the case of SLu-derived mouse tumor cells, tumors also developed in the immunocompetent BALB/c mice using 1×10⁶ to 5×10⁶ tumor cells for SC transplantation.     

In vivo insulin action in adrenodemedullated rats after voluntary running

The purpose of this study was to estimate the influence of epinephrine on in vivo insulin sensitivity and responsiveness after voluntary running. Wistar rats that had previously undergone adrenodemedullation or sham-operation were kept in a sedentary state or trained over a 4 week period. An euglycemic insulin clamp study was performed on adrenodemedullated sedentary rats (ADMX), adrenodemedullated voluntary running rats (ADMX-T), sham-operated voluntary running rats (SHAM-T), and control rats (C) at 18 h after the last bout of exercise. The insulin infusion rate was 3.0, 6.0, and 303.0 mU/(kg min), respectively. The blood glucose concentration was maintained constant at basal levels. Metabolic clearance rate of glucose (MCR) was calculated as an index of whole-body insulin action. In the presence of physiological hyperinsulinemia (an insulin infusion rate of 6.0 mU/(kg min)), MCR (ml/(kg min)) was significantly higher in ADMX-T rats (31.2 +/- 2.0) than in ADMX rats (19.8 +/- 0.8, P<0.001) and SHAM-T rats (23.8 +/- 0.8, P<0.05). Also, the MCR values of SHAM-T and ADMX rats were significantly (P<0.001, and P<0.05, respectively) greater than that of C rats (12.7 +/- 0.4). At maximal hyperinsulinemia (an insulin infusion rate of 303.0 mU/(kg min)), there was no difference of MCR between ADMX-T rats (49.8 +/- 4.3) and C rats (38.2 +/- 2.2). The GDR values of SHAM-T rats (43.5 +/- 3.7) and ADMX rats (43.5 +/- 2.1) were also not different from that of C rats. These results provide indirect evidence that epinephrine is one of factors that suppresses increased insulin sensitivity after physical training, although it seems to have no significant influence on insulin responsiveness.     

In vivo-like growth of human tumors in vitro.

We show that diverse human tumors obtained directly from surgery or biopsy can grow at high frequency in vitro for long periods of time and still maintain many of their in vivo properties. The in vivo properties maintained in vitro include three-dimensional growth; maintenance of tissue organization and structure, including changes associated with oncogenic transformation; retention of differentiated function; tumorigenicity; and the growth of multiple types of cells from a single tumor.     

In vivo antagonism with zidovudine plus stavudine combination therapy

Human immunodeficiency virus (HIV)-infected subjects receiving zidovudine were randomized either to add stavudine (d4T) or didanosine (ddI) to their current regimen or to switch to ddI or d4T monotherapy. After 16 weeks of therapy, the mean reduction in HIV RNA from baseline was 0.14 log(10) copies/mL in patients receiving d4T or zidovudine plus d4T. In subjects receiving ddI or ddI plus zidovudine, reductions were 0.39 and 0.56 log(10), respectively. CD4 cell counts remained stable or showed modest increases in all arms except the zidovudine plus d4T arm. Patients receiving zidovudine plus d4T showed progressive declines in CD4 cell counts with a median of 22 cells/mm(3) below baseline by 16 weeks. Examination of intracellular levels of d4T-triphosphate in 6 subjects was consistent with previous in vitro studies demonstrating pharmacologic antagonism between zidovudine and d4T. Analysis of these data suggests that zidovudine and d4T should not be prescribed in combination and that ddI provides greater antiviral activity than d4T in zidovudine-treated patients.     

In vitro and in vivo growth hormone responsiveness to growth hormone-releasing factor in male and female Zucker rats

In order to determine whether there is an anomaly in the pituitary responsiveness to growth hormone (GH)-releasing factor in the genetically obese rat, we examined the in vitro and in vivo effects of rGRF(1-29)NH2 (GRF) on GH release in male and female Zucker rats. The effect of increasing GRF concentrations (1.56, 6.25, 12.5 25 and 50 pM) was first tested on GH release from freshly perifused anterior pituitary cells. In both sexes, the GH response per one pituitary equivalent to each GRF concentration tested was reduced in the obese group. However, when GH release was expressed as a percent of initial cell GH content, there was no difference between the lean and the obese groups. Furthermore, under pentobarbital anesthesia, GRF was injected intravenously at two consecutive doses of 0.8 and 4.0 microgram/kg body weight in obese and lean animals. In both sexes, the GH response to each dose of GRF tested was decreased in the obese group. Basal serum GH concentrations were similar in male and female obese rats compared to their respective lean siblings. In conclusion, this study demonstrates a decrease of the in vitro and in vivo pituitary response to GRF in the obese Zucker rat, suggesting a possible secondary defect at the pituitary level.     

Endothelin antagonism improves hepatic insulin sensitivity associated with insulin signaling in Zucker fatty rats

In the present study, we investigated the effects of long-term treatment with the endothelin (ET) antagonist atrasentan, an ET(A)-selective antagonist, on whole body glucose metabolism and insulin signaling in a commonly used model of insulin resistance, the Zucker fatty rat. Zucker lean and fatty rats were maintained for 6 weeks on either control or atrasentan-treated water. Euglycemic-hyperinsulinemic clamps (4 mU/kg per minute) were performed at the end of the 6-week treatment on a subset of rats (n=10/treatment). In another subset (n=5/treatment), an insulin tolerance test was performed; liver and muscle tissues were harvested 10 minutes following the challenge for further analysis. Results of the clamps demonstrated that long-term atrasentan treatment significantly increased whole body glucose metabolism in fatty rats compared with vehicle control subjects. Insulin-induced insulin receptor substrate 1 tyrosine and protein kinase B serine phosphorylation were significantly reduced in the liver and muscle of fatty animals compared with their lean littermates. This reduction was overcome with atrasentan treatment in the liver but not in the muscle. There was no difference between lean and fatty animals, however, in insulin receptor substrate 1 and protein kinase B protein expression in the liver and muscle and no effect by atrasentan. In contrast, expression of the regulatory subunit of PI-3 kinase (p85alpha) was significantly increased in the liver but not in the muscle of fatty animals compared with their lean littermates and this was normalized to levels of lean animals with atrasentan treatment. These findings indicate that long-standing ET antagonism improves whole body glucose metabolism in Zucker fatty rats through improvements in insulin signaling in the liver. These results indicate that therapeutic ET antagonism may assist in correcting the insulin-resistant state.     

In vitro insulin effect on acetylcholine esterase of erythrocyte membranes of normal and diabetic rats

Summary Alloxan induced diabetes in rats was associated with a significant reduction in the acetylcholine esterase activity of the erythrocyte membrane. Preincubation of these membranes with insulin caused a rapid but transient stimulation of this enzyme activity in both normal and diabetic rats, the effect being more marked in the latter group. CDRI Communication No. 3721.     

Effects of insulin in vitro in the fatty tissue of rats with alloxan diabetes after insulin treatment or administration of insulin-like growth factors from human serum

Insulin insufficiency in rats caused by alloxan, results in a decrease in the sensitivity of the fatty tissue to insulin determined by glucose oxidation and synthesis of lipids manifested in a decrease in a maximum response of the tissue to insulin. Multiple injection of insulin as well as of partially purified total fraction of insulinoid growth factors causes an increase in a maximum response of the fatty tissue to insulin and a right-hand shift of dose-response curves. The total effect of the injection of insulin or partially purified growth factors is expressed in elevated sensitivity of the fatty tissue to insulin.