Effect of Long-Term Intake of Y^(3+) in Drinking Water on Gene Expression in Brains of Rats

The rats were fed with water dissolved Y^3＋ at different levels （0, 53.4, 5340 mg·L^-1） for 7 months. The gene expression in brain tissue was detected with oligonucleotide microarray. The results show that, compared to the control, 789 genes express differentially, 507 over-expressed genes and 282 under-expressed genes in the high-dose group （5340 mg· L^-1）, of which, most were related to cell receptor, cell signal and transmission, and ionic passage. 44 genes were found to express differentially including 32 over-expressed genes and 12 under-expressed genes in the low-dose group （53. 40 mg· L^-1）, of which, most were related to cell skeleton and movement, immunity, and DNA binding protein. These resuits suggest that Y^3. can change the expression of some genes, which may be responsible for the toxicity of rare earths on learning and memory.

HBV X Gene Transfection Upregulates IL-1β and IL-6 Gene Expression and Induces Rat Glomerular Mesangial Cell Proliferation

The X gene of HBV encodes a 17-kD protein, termed HBx, which has been shown to function as a transcriptional trans-activator of a variety of viral and cellular promoter/enhancer elements. The aim of this study was to investigate the effect of HBx on gene expression of interleukin （IL）-1β and IL-6, and proliferation of rat mesangial cells in vitro. The X gene of HBV was amplified by PCR assay, and inserted into the eukaryotic expression vector pCI-neo. The structure of recombinant pCI-neo-X plasmid was proved by restrict endonuclease digestion and sequencing analysis. pCI-neo-X was transfected into cultured rat mesangial cell line in vitro via liposome. HBx expression in transfected mesangial cells was detected by Western blot. The IL-1β and IL-6 mRNA expression in those cells was assayed by semiquantitative RT-PCR. Mesangial cell proliferation was tested by MTT. The results showed that HBx was obviously expressed in cultured mesangial cell line at 36th and 48th h after transfection. The expression of IL-1β and IL-6 mRNA was simultaneously increased. The cell proliferation was also obvious at the same time. It was concluded that HBx gene transfection could induce IL-1β and IL-6 gene expression and mesangial cell proliferation. HBx may play a critical role in mesangial cell proliferation through upregulation of the IL-1β and IL-6 gene expression.

Attenuation of Nitric Oxide Synthase Gene Expression in Rat Lung Induced by Hypoxia

In order to investigate the role of nitric oxide(NO) in pathogenesis of hypoxic pulmonary hypertension(HPH), the mean pulmonary arterial pressure(mPAP), mRNA expression of NO synthase(NOS) in lung tissues, cGMP levels, and their relationships were studied in rats exposed to hypoxia from 8 h to 28days. The results showed that mPAP began to increase in animals exposed to 10 % O2 for 8 h. Moreover, the longer the exposure, the higher the mPAP.Northern blot analysis and dot blot hybridization indicated that mRNA expression of NO in lung tissues of hypoxic rats tended to decrease with exposure days, but that of β-actin which acted as a control did not alter. The cGMP levels of plasma and lung tissues in hypoxic rats also inclined to be lower with exposure days. A marked negative correlations between the changes of cGMP levels and those of mPAP were found. It was suggested that mRNA expression of NOS gene was attenuated in hypoxic lung tissues, which may be one of important pathogenetic mechanisms of HPH.

Preliminary study on androgen dependence of calcitonin gene-related peptide in rat penis

Aim: To study the androgen dependence of the neurotransmitter, calcitonin gene-related peptide (CGRP) in rat penis. Methods: Forty-four Sprague-Dawley rats were randomly divided into Group A (intact controls), Group B (castrated) and Group C (gavaged with finasteride 4.5 mg·kg^(-1).day^(-1)). Four and ten weeks later respectively, half of rats in each group were anaesthetized. Blood samples were taken for the measurement of serum testosterone and dihydrotestosterone (DHT) by means of radioimmunoassay. Penile samples were harvested for the investigation of calcitonin gene related peptide (CGRP)-immunoreactive nerve fibers with immunohistochemistry. The computer-assisted imaging analysis system was applied to calculate the area proportion of the CGRP-positive nerve fibers (CGRP-PNF) in each group. Results: 1) Both 4 and 10 weeks later, testosterone and DHT levels in Group B decreased significantly compared with those in Group A, (P < 0.05, P < 0.01, respectively); DHT level in Group C was also significantly decreased in comparison with that in Group A for both 4- and 10- week animals (P < 0.05); 2) There was no significant differences in area proportion of CGRP-PNF among Groups A, B and C 4 weeks after treatments (P > 0.05); However, 10 weeks later, the proportion of CGRP-PNF in Groups B and C was significantly less than that in Group A (P < 0.01);3) The proportion of CGRP-PNF of 4-week animals in Groups B and C was significantly higher than that of 10-week animals (P < 0.05). Conclusion: The expression of neurotransmitter, CGRP may depend on androgens, including testosterone and DHT in rat penis.

Effect of adenovirus-mediated gene transfer of Olig1 on oligodendrocyte differentiation and remyelination in a rat model of focal cerebral ischemia

BACKGROUND： The transcription factor Oligl is required for oligodendrocyte maturation and demyelinated lesion repair, and is a key regulator of myelinogenesis following ischemia. OBJECTIVE： To examine the efficacy of intraventricular injection of a recombinant adenovirus-expressing Oligl gene （Ad5-Oligl-eGFP） on oligodendrocyte maturation and myelin repair following focal cerebral ischemia. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Department of Neurology, Beijing Friendship Hospital Affiliated to Capital Medical University from January 2007 to March 2008. MATERIALS： Adenovirus and a recombinant adenovirus containing Oiigl gene （Ad5-Oligl） were provided by Vector Gene Technology, China. METHODS： All 50 rats were induced by middle cerebral artery occlusion. A total of 46 rats were successfully induced and were subsequently randomly assigned to a adenovirus （Ad5） group and recombinant adenovirus-expression Oligl gene （Ad5-Oligl） group, with 23 rats per group. One day after middle cerebral artery occlusion, either Ad5-Oligl-eGFP or Ad5-eGFP （10 μL, 2.3 ×10^11/mL） was injected into the lateral ventricle on the ischemic hemisphere. MAIN OUTCOME MEASURES： Adenovirus-mediated Oligl gene expression in vitro and in vivo was measured by reverse transcription-polymerase chain reaction and immunofluorescence, respectively. Myelin basic protein （MBP） levels were evaluated by Western Blot, immunostaining, and electron microscopy. RESULTS： Exogenous Oligl expression was measured at the periventricular zone of the lateral ventricle 1 day after Ad5-Oligl injection. In the Ad5-Oligl-treated group, MBP protein levels and average intensity of MBP-immunoreactivity （-ir） increased 28 days after middle cerebral artery occlusion, compared with the control group （P 〈 0.01, P 〈 0.05）. Furthermore, myelinated axonal numbers markedly increased following Ad5-Oligl treatment. CONCLUSION： The present data suggested that Ad5-Oligl gene therapy increased MBP expression and the number of remyelinating axons following cerebral ischemia.

The effect of microgene pSVPoMcat to modify Schwann cell on GAP -43 expression after spinal cord injury in adult rats

Objective To study the effect of microgene pSVP oMcat implanted to modify schwann cell on growth associated protein -43(GAP -43)expression after spinal cord injury in adult rats.Method Hemisected of the T8segment of the sp inal cord was performed for all the experiment rats.The rats were randomly divided into three groups as follows:Group Awith microgene pSVPoMca t implanted to genetically modify SC;Group B with SC implanted ;Group C with hemisection of the spinal cord o nly.The changes of expression of GAP-43in spinal cord were observed by immunochemistry with antibodies against GAP -43.Simultaneous,the combined behavioral scores(CBS)was measured.Result There were not any different (P >0.05)among the three groups in first week a nd 12week.There were significant di ffeence(P <0.05)among three groups in 2nd,8th,and more dxpression of GAP -43at the 2nd week in gr oup A.The neurofunctional recovery was best in group A.Conclusion The microgene pSVPoMcat implanted t o modify schwann cell can promote the expression of GAP -43in spinal cord a nd func-tional recovery in adults rats after SCI.

Inhibiting effect of antisense oligonucleotides phosphorthioate on gene expression of TIMP-1 in rat liver fibrosis

AIM: To observe the inhibition of antisense oligonucleotides (asON) phosphorthioate to the tissue inhibitors metalloproteinase-1 (TIMP-1) gene and protein expression in the liver tissue of immunologically induced hepatic fibrosis rats. The possibility of reversing hepatic fibrosis through gene therapy was observed. METHODS: Human serum albumin (HSA) was used to attack rats, as hepatic fibrosis model, in which asONs were used to block the gene and protein expressing TIMP-1. According to the analysis of modulator, structure protein, coding series of TIMP-1 genome, we designed four different asONs. These asONs were injected into the hepatic fibrosis models through coccygeal vein. The results was observed by RT-PCR for measuring TIMP-1 mRNA expression, immunohistochemistry and in situ hybridization for collagen I, II, special staining of collagen fiber, and electron microscopic examination. RESULTS: Hepatic fibrosis could last within 363 days in our modified model. The expressing level of TIMP-1 was high during hepatic fibrosis process. It has been proved by the immunohistochemical and the electron microscopic examination that the asON phosphorthioate of TIMP-1 could exactly express in vivo. The effect of colchicine was demonstrated to inhibit the expressing level of mRNA and the content of collagen I, III in the liver of experimental hepatic fibrosis rats. However, the electron microscopy research and the pathologic grading of hepatic fibrosis showed that there was no significant difference between the treatment group and the model group (P】 0.05). CONCLUSION: The experimental rat model of hepatic fibrosis is one of the preferable models to estimate the curative effect of anti-hepatic fibrosis drugs. The asON phosphorthioate of TIMP-1 could block the gene and protein expression of TIMP-1 in the liver of experimental hepatic fibrosis rats at the mRNA level. It is possible to reverse hepatic fibrosis, and it is expected to study a new drug of antihepatic fibrosis on the genetic level. Colchicine has very limited therapeutic effect on hepatic fibrosis, furthermore, its toxicity and side effects are obvious.

Amelioration of carbon tetrachloride-induced cirrhosis and portal hypertension in rat using adenoviral gene transfer of Akt

AIM:To investigate whether a virus constitutively expressing active Akt is useful to prevent cirrhosis induced by carbon tetrachloride(CCl4).METHODS:Using cre-loxp technique,we created an Ad-myr-HA-Akt virus,in which Akt is labeled by a HA tag and its expression is driven by myr promoter.Further,through measuring enzyme levels and histological structure,we determined the efficacy of this Ad-myrHA-Akt virus in inhibiting the development of cirrhosis induced by CCl4in rats.Lastly,using western blotting,we examined the expression levels and/or phosphorylation status of Akt,apoptotic mediators,endothelial nitric oxide synthase(eNOS),and markers for hepatic stellate cells activation to understand the underlying mechanisms of protective role of this virus.RESULTS:The Ad-myr-HA-Akt virus was confirmed using polymerase chain reaction amplification of inserted Akt gene and sequencing for full length of inserted fragment,which was consistent with the sequence reported in the GenBank.The concentrations of Admyr-HA-Akt and adenoviral enhanced green fluorescent protein(Ad-EGFP)virus used in the current study were5.5×1011vp/mL.The portal vein diameter,peak velocity of blood flow,portal blood flow and congestion index were significantly increased in untreated,saline and Ad-EGFP cirrhosis groups when compared to normal control after the virus was introduced to animal through tail veil injection.In contrast,these parameters in the Akt cirrhosis group were comparable to normal control group.Compared to the normal control,the liver function(Alanine aminotransferase,Aspartate aminotransferase and Albumin)was significantly impaired in the untreated,saline and Ad-EGFP cirrhosis groups.The Akt cirrhosis group showed significant improvement of liver function when compared to the untreated,saline and Ad-EGFP cirrhosis groups.The Hyp level and portal vein pressure in Akt cirrhosis groups were also significantly lower than other cirrhosis groups.The results of HE and Van Gieson staining indicated that Akt group has better preservation of histological structure and less fibrosis than other cirrhosis groups.The percentage of apoptotic cell was greatly less in Akt cirrhosis group than in other cirrhosis groups.Akt group showed positive HA tag and an increased level of phosphorylated Akt as well as decreased levels of Fas.In contrast,Caspase-3 and Caspase-9 levels in Akt group were significantly lower than other cirrhosis groups.Noticeable decrease of DR5 andα-SMA and increase of phosphorylated eNOS were observed in the Akt group when compared to other cirrhosis groups.The NO level in liver was significantly higher in Akt group than other cirrhosis groups,which was consistent with the level of phosphorylated eNOS in these groups.CONCLUSION:This study suggest that Ad-myr-HA-Akt virus is a useful tool to prevent CCl4-induced cirrhosis in rat model and Akt pathway may be a therapeutic target for human cirrhosis.

Changes in 5α-reductase (type 2) gene expression in epididymis of puberty diabetic rats

The changes in 5α-reductase (type 2 ) gene expression in the epi-didymis of puberty diabetic rats were studied by the Northern blot and Dotblot method. Rats were divided into 3 groups: the control group (C), thediabetic group (D), and the diabetic group with insulin treatment (DI).Results: The Northern blot intensity of the caput epididymis in Group D is

Expression of vasa Gene in Development Process of Germ Cells in Male Rat

In order to explore the role of vasa gene in the development of germ cells after gonad differentiation in male rats,the expression of vasa mRNA and vasa protein in 17.5-day-old fetal rats and neonatal rats was detected by real-time fluorescent quantitative PCR and immunohistochemistry method.The results showed that the expression of vasa mRNA was detected in the testis tissue of 17.5-day-old fetal rats and neonatal rats,and the expression of vasa mRNA in testis of neonatal rats was high than that in fetal rats.The expression of vasa protein was detected in neonatal rats,but it was not found in fetal rats.In conclusion,vasa gene plays an important role in the development of germ cells.However,as a marker,it can only be used to label all kinds of germ cells after formation of prespermatogonia.

Generation and characterization of a novel CYP3A1/2 double knockout rat model using CRISPR-Cas9 system

OBJECTIVE Cytochrome P450(CYP)3A accounts for nearly 30%of total CYP enzymes in human liver and participates in the metabolism of over 50%of clinical drugs.CYP3A also plays an important role in the chemical metabolism,toxicity,and carcinogenicity.New animal models are needed to investigate CYP3A functions.METHODS The CRISPR-Cas9 technology was used to generate Cyp3a1/2 double knockout rat model.The absence of Cyp3a1/2 expression was evaluated through PCR and immunostaining.Metabolic studies of the CYP3A substrates midazolam and nifedipine both in vitro and in vivo were conducted to verify that CYP3A1/2 was functional y inactive in KO rats.In addition,compensatory up-regulation of other P450 genes in Cyp3a1/2 KO rats was detected.RESULTS The Cyp3a1/2 double KO rats were viable and fertile,and had no obvious physiological abnormities.Compared with the wild-type(WT)rat,Cyp3a1/2 expression was completely absent in the liver of the KO rat.In vitro and in vivo metabolic studies of the CYP3A1/2 substrates indicated that CYP3A1/2 was functionally inactive in double KO rats.CONCLUSION The Cyp3a1/2 double KO rat model was successfully generated and characterized.The Cyp3a1/2 KO rats as a novel rodent animal model will be a valuable tool for the study of the physiological and pharmacological roles of CYP3A,and determining whether the absence of CYP3A results in non-CYP mediated metabolism or metabolism by other CYP isoforms.

Study on Long-Terms Survival and Gene Expression of pSVPoMcat Genetically Modified Schwann Cell in the Rat Spinal Cord Injure

Objective To observe the survival and gene expression of genetically modified schwann cell (SC) with pSVPoMcat in the rat spinal cord.Methods The experimental animals were divided into three groups randomly, that is, pSVPoMcat modified SC implantation group(Group A),highly purified SC implantation group(Group B),and the controlled group (Group C). After operation, the survival time for each group was 2, 4, 8, 12 weeks respectively, 10 rats once for each group, then the section of spinal cord grafted were checked by immunocytochemical staining of S－100 protein, myelin basic protein(MBP) and in situ hybridization.Results After operation for 2, 4. 8. 12 weeks, Group A’ S－100, MBP staining cells and ISH cells were respectively (82.3± 6.07)％ , (77.8± 12.3)％ , (67.8± 8.6)％； (81.2± 5.2)％ , (76.3± 11.8)％ , (68.4± 8.3)％； (80.7± 5.6)％ , (75.4± 11.6)％ ,(66.4± 8.2)％； (81.6± 6.2)％ , (75.8± 9.5)％ , (63.3± 8.02)％ ,(P<0.05). Among them, We can find that myelin sheath emerges in ISH positive cells Group B’S S－100 staining cells are (66.3± 6.8)％ , (40.2± 11.2)％ , (10.8± 2.6)％ , (3.2± 0.6)％ ,(P< 0.001), but no MBP staining cells and ISH cells are found. And in Group C, no positive cells are found at all.Conclusion pSVPoMcat genetically modified SC can survive for a long time and express gene in intraspinal injured and help to formation myelin of spinal cord injured (SCI) which provide a feasible way to gene care to spinal injury.

Analysis of candidate genes of QTL and chromosomal regions for essential hypertension in the rat model

This is an in silico analysis of quantitative trait loci (QTLs), genes, polymorphisms, and chromosomal regions regulating hypertension in the rat genome. Utilizing PGmapper, a program that matches phenoltypes to genes, we identified 266 essential hypertension-associated genes (HyperA), and 83 of these genes contain known hypertension-associated polymorphisms (HyperAP). The majority of HyperAP have been reported in recent years. Surprisingly, only a few of these HyperAP genes have been investigated for their candidacy as the QTL for hypertension. The frequency of candidate genes within peak regions of the QTL is higher than the rest of the QTL region. We also found that QTL located in both gene-rich regions and gene-rich chromosomes contained the most candidate genes. However, the number of candidate genes within a peak region is not associated with the number of total genes in a QTL region. This data could not only facilitate a more rapid and comprehensive identification for the causal genes underlying hypertension in rats, but also provides new insights into genomic structure in regulation of hypertension.

Cloning and Expression of Osteonectin Gene from Rats

Total cellular RNA was extracted from the osteoblast cells of uewborn rats＇ calvarial bones, and the cDNA containing open-reading frame of osteonectin was amplified by reverse transcription polymerase chain reaction （ RT- PCR ）. The obtained product was named On. The On fragment was inserted into pBT- T vector. Then, the On was subdoued, in-frame fused to 3＇ -end of the GST gene of the prokaryotic expression vector pGEX-KG, and the resulting recombinant plasmid was transformed into E. coli BL21 （DE3） pLysS competent cells. A 60 kD fusion protein was expressed after IPTG induction. The On fragment was sequenced, and the sequencing result shows that it shares 99.8% homology with the sequence published in GenBank. The On SDSPAGE analysis exhibits that the On was expressed with the GST gene. There is 10% fused protein in the total E. coli proteins, and the fasion protein is a soluble protein. These experimental results imply that On from Wistar rats was cloned suecessfully and expressed efficiently.

PROMOTION OF CHEMICAL CARCINOGENESIS AND P53 EXPRESSION BY REDUCTION OF SUPEROXIDE DISMUTASE ACTIVITY IN THE LUNG OF RAT IN VIVO

In the present experiment,an inhibitor of superoxide dismutase(SOD),diethyldithiocarbamate(DETC),was used to decrease SOD activity for the observation of the relation between SOD activity and carcinogenesis and the expression of P53 protein in vivo.144 Wistar rats were used for the Present study.The results showed that the SOD activity reduction by DETC resulted markedly in the promotion of the carcinogenesis and the expression of P53 protein in the lung tissues,but the increase of SOD activity by the addition of plus SOD inhibited the pathological changes significantly.The frequency of the pathological lesions and Positive P53 expression are 36/42 and 8/42 in the animals without DETC and SOD:16/52 and 4/52 in the animals with SOD and 46/50 and 26/50 in the animals with DETC respectively.The results reported in this Paper suggest that:(1) the decrease of SOD activity enhanced the carcinogenesis induced by chemical carcinogen;(2) P53 gene may be associated with the process of tumorigenesis;and(3) at the same time the abnormal expression of P53 protein may be associated with the transition from premalignant lesions to carcinoma.

Gene expression and histopathological evaluation of thiamine pyrophosphate on optic neuropathy induced with ethambutol in rats

AIM： To compare the effects of thiamine pyrophosphate（TPP） and thiamine（TM） in oxidative optic neuropathy in rats induced by ethambutol.METHODS： The animals were divided into four groups：a control group（CG）,an ethambutol control（ETC） group,TM plus ethambutol group（TMG）,and TPP plus ethambutol group（TPPG）.One hour after intraperitoneal administration of TM 20 mg/kg to the TMG group and TPP 20 mg/kg to TPPG group,30 mg/kg ethambutol was given via gavage to all the groups but the CG.This procedure was repeated once daily for 90 d.After that period,all rats were exposed to high levels of anaesthesia in order to investigate the gene expression of malondialdehyde and glutathione in removed optic nerve tissue and histopathologically to examine these tissues. RESULTS： Malondialdehyde gene expression significantly increased,whereas glutathione gene expression significantly decreased in the ETC group compared to the CG.TM could not prevent the increase of malondialdehyde gene expression and the decrease of glutathione,while TPP significantly could suppress.Histopathologically,significant vacuolization in the opticnerve,single-cell necrosis in the glial cells,and a decrease in oligodendrocytes were observed in the ETC group.Vacuolization in the optic nerve,a decrease in oligodendrocytes and single-cell necrosis were found in the TMG group,while no pathological finding was observed in the TPPG group except for mild vacuolization.CONCLUSION： TPP protects the optic nerve against the ethambutol-induced toxicity but TM does not.TPP can be beneficial in prophilaxis of optic neuropathy in ethambutol therapy.

Retrovirus vector transfection of rat insulin gene into pancreas decrease blood glucose of diabetic rat

Human and animal diabetes mellitus were controlled by a dietary treatment supplemented with either a sulfonylurea drug or insulin injection. Insulin injections were inconvenient and the hypoglycemia induced by insulin-overdose could be fatal. Sulfonylurea drugs were administered orally, however, do not typically provide satisfactory control of blood glucose as a starting treatment in 25% - 30% patients. Therefore, it was imperative to develop a method for the control of human and animal diabetes mellitus. Recently, insulin gene transferred and expressed in non-pancreatic cells as a means for the treatment of diabetes was developed rapidly in the expanding gene therapy. Retrovirus, lentivirus, adenovirus, adenoassociated virus and herpes simplex had been used as viral vectors, and the constructed viral-insulin gene was successfully transferred into diabetic rat cells. A gene, containing promoter, enhancer and rat type I insulin gene (a-chain, b-chain and signal peptide), was constructed into a retrovirus vector in the study. The constructed viral-insulin gene was transferred into mouse fibroblast cell. The insulin concentration in 3-day cultured mouse fibroblast cells was 4806.35 ± 53.72 pg/ml. The insulin concentration for the viral vector containing enhancer and promoter of rat insulin gene was higher than the vector containing only insulin gene by a 61% increase in the cultured mouse fibroblast cells. The enhancer and promoter activity of rat insulin gene would be an important determinant for the expression of insulin gene. The secreted amount of insulin by retrovirus vector contained enhancer/promoter gene in this study could achieve as high concentrations (4806.35 ± 53.72 pg/ml) as the insulin injection therapy. Blood glouse decreased sig- nificantly for at last 10 days demonstrated that transfection, direction injection of viral-insulin gene into pancreas of diabetic rat, was successful. These studies suggest that the retrovirus vector might be used to transfer the insulin gene in vitro and in vivo.

Study on the Regulation of Bcl-2 Gene on Rat Spermatogenic Cells Apoptosis in Transcription Level

Objective To detect the change of Bcl 2 gene expression in the apopototic process of spermatogenic cells in rat with vasoligation and vasostomy, and to find out the relationship between the transcription of Bcl 2 and the apoptosis of spermatognic cells Materials & Methods Sixty adult male Sprague Dawley rats in 3 groups were operated with vasoligation and vasostomy. Then hybridization in situ with hypersensitive Bcl 2 RNA probe was used to detect the change of Bcl 2 mRNA. Results The transcription of Bcl 2 gene in spermatogenic cells was obviously inhibited in the vasoligation group compared with that in the control group (P<0.05), and the transcription in the vasostomy group showed no difference from that of the control group. Conclusion Bcl 2 gene has an anti apoptotic effect in rats with vasostomy, and there was a transcriptional regulation of Bcl 2 gene in rat spermatogenic cell during the period of pre vasoligation to post vasoligation and to post vasosotomy.

1,25-Dihydroxyvitamin D_(3) effects on the regulation of the insulin receptor gene in the hind limb muscle and heart of streptozotocin-induced diabetic rats

In the present study, we examine the effects of the treatment with 1,25-dihydroxyvitamin D3 [150 IU/Kg (3.75 μg/Kg) once a day, for 15 days] to non-diabetic and streptozotocin-induced diabetic rats. The results indicate that treatment with 1,25-dihydroxyvitamin D3 had minor effects in non-diabetic rats. The same treatment in streptozotocin-induced diabetic rats, although it did not correct the hyperglycemia and hypoinsulinemia induced by the diabetes, caused other actions that could mean beneficial effects on the amelioration of diabetes e.g., it avoided body weight loss, increased calcium and phosphorus plasma levels, and corrected the over-expression of the insulin receptor mRNA species of 9.5 and 7.5 Kb present in the hind limb muscle and heart of these animals. These genomic 1,25-dihydroxyvitamin D3 effects could involve transcriptional mechanisms of repression mediated by vitamin D response elements in the rat insulin receptor gene promoter. Using computer analysis of this promoter, we propose the -249/-235 bp VDRE (5’GGGTGACCCGGGGTT3’) with a pyrimidine (T) in the (+7) position of the3’half-site as the best candidate for negative control by 1,25-dihydroxy-vitamin D3. In addition, posttranscriptional mechanisms of regulation could also be implicated. Thus, computer inspection of the5’untranslated region of the rat insulin receptor pre-mRNA indicated the presence of a virtual internal ribosome entry segment whereas the computer inspection of the3’untranslated region localized various destabilizing sequences, including various AU-rich elements. We propose that through these virtual cis-regulatory sequences, 1,25-dihydroxyvitamin D3 could control the translation and stability of insulin receptor mRNA species in the hind limb muscle and heart of diabetic rats.

High yield reproducible rat model recapitulating human Barrett's carcinogenesis

AIM To efficiently replicate the biology and pathogenesis of human esophageal adenocarcinoma(EAC) using the modified Levrat model of end-to-side esophagojejunostomy. METHODS End-to-side esophagojejunostomy was performed on rats to induce gastroduodenoesophageal reflux to develop EAC. Animals were randomly selected and serially euthanized at 10(n = 6),17(n = 8),24(n = 9),31(n = 6),38(n = 6),and 40(n = 6) wk postoperatively. The esophagi were harvested for downstream histopathology and gene expression. Histological evaluation wascompleted to determine respective rates of carcinogenic development. Quantitative reverse transcriptionpolymerase chain reaction was performed to determine gene expression levels of MUC2,CK19,and CK20,and results were compared to determine significant differences throughout disease progression stages.RESULTS The overall study mortality was 15%. Causes of mortality included anastomotic leak,gastrointestinal hemorrhage,stomach ulcer perforation,respiratory infection secondary to aspiration,and obstruction due to tumor or late anastomotic stricture. 10 wk following surgery,100% of animals presented with esophagitis. Barrett's esophagus(BE) was first observed at 10 wk,and was present in 100% of animals by 17 wk. Dysplasia was confirmed in 87.5% of animals at 17 wk,and increased to 100% by 31 wk. EAC was first observed in 44.4% of animals at 24 wk and increased to 100% by 40 wk. In addition,two animals at 38-40 wk post-surgery had confirmed macro-metastases in the lung/liver and small intestine,respectively. MUC2 gene expression was progressively down-regulated from BE to dysplasia to EAC. Both CK19 and CK20 gene expression significantly increased in a stepwise manner from esophagitis to EAC. CONCLUSION Esophagojejunostomy was successfully replicated in rats with low mortality and a high tumor burden,which may facilitate broader adoption to study EAC development,progression,and therapeutics.