Rdh13 deficiency weakens carbon tetrachloride-induced liver injury by regulating Spot14 and Cyp2e1 expression levels / 医学前沿

Mitochondrion-localized retinol dehydrogenase 13 (Rdh13) is a short-chain dehydrogenase/reductase involved in vitamin A metabolism in both humans and mice. We previously generated Rdh13 knockout mice and showed that Rdh13 deficiency causes severe acute retinal light damage. In this study, considering that Rdh13 is highly expressed in mouse liver, we further evaluated the potential effect of Rdh13 on liver injury induced by carbon tetrachloride (CCl). Although Rdh13 deficiency showed no significant effect on liver histology and physiological functions under regular culture, the Rdh13 mice displayed an attenuated response to CCl-induced liver injury. Their livers also exhibited less histological changes and contained lower levels of liver-related metabolism enzymes compared with the livers of wild-type (WT) mice. Furthermore, the Rdh13 mice had Rdh13 deficiency and thus their liver cells were protected from apoptosis, and the quantity of their proliferative cells became lower than that in WTafter CCl exposure. The ablation of Rdh13 gene decreased the expression levels of thyroid hormone-inducible nuclear protein 14 (Spot14) and cytochrome P450 (Cyp2e1) in the liver, especially after CCl treatment for 48 h. These data suggested that the alleviated liver damage induced by CCl in Rdh13 mice was caused by Cyp2e1 enzymes, which promoted reductive CCl metabolism by altering the status of thyroxine metabolism. This result further implicated Rdh13 as a potential drug target in preventing chemically induced liver injury.

Practice of course construction of medical molecular genetics for postgraduates and its sugges-tion / 中华医学教育探索杂志

To improve the quality of postgraduate students,school of medicine of Shanghai Jiao Tong University have offered medical molecular genetics course to postgraduates and obtained good teaching efficiency. More than ten professors gave lectures on the academic foreland,leading students to find and solve academic problems and training practical and innovative ability of students. Accord-ing to the questionnaires of evaluation of the course and suggestions from postgraduates in the recent two years,some recommendations for the further teaching reform were proposed,such as using multi-plex teaching models,strengthening communication for teaching information with the students,em-ploying comprehensive evaluation methods and strengthening coordination and management of the course.

Generation of factor VIII gene knockout mouse by tetraploid embryo complementation technology / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>Factor VIII( FVIII) gene knockout mouse model was established for further study on the treatment of hemophilia A.</p><p><b>METHODS</b>Exons 16-19 of the mouse FVIII gene were knocked out by ET clone, ES homologous recombination and tetraploid embryo compensation technology. PCR, reverse transcriptase-PCR(RT-PCR) and immunohistochemistry were used to detect the transcription and translation pattern of FVIII. The phenotype of the knockout mice was analyzed by examining the activated partial thromboplastin time (APTT) and FVIII activity (FVIII:C).</p><p><b>RESULTS</b>PCR, RT-PCR and immunohistochemistry confirmed that FVIII was deficient in the FVIII gene knockout mouse. The APTT results showed that FVIII-deficient mouse plasma had a prolonged clotting time compared to normal mouse plasma. The FVIII:C in heterozygous, hemizygous and homozygous mice was 80%, 8% and 10% of that in normal mice, respectively.</p><p><b>CONCLUSION</b>The phenotype of the FVIII gene knockout mouse appears grossly similar to that of human with hemophilia A. Establishment of this model may promote the development of new technologies of treatment to hemophilia A.</p>

High-bone-turnover Osteoporosis and Aortic Calcification in Opg Knockout Mice / 生物化学与生物物理进展

Bone turnover is regulated by local concentrations of cytokines such as osteoprotegerin (OPG) and receptor activator of nuclear factor kappaB ligand (RANKL). To explore the in vivo biological function of Opg and the mechanism of osteoporosis due to deficiency of Opg, Opg knockout mice have been generated through homologous recombination. Opg-/- mice exhibit a sharply decrease in bone density and strength as expected. The number of osteoclasts in Opg-/- mice significantly increases. Morphologically, osteoclasts appear more cuboidal in shape in Opg-/- mice than those of wt mice, suggesting that active osteoclastogenesis occurs in the absence of Opg. In consistent with this finding, an increase of osteoblast activity was also observed with accelerated mineral accumulation rate by histomorphometric measurement and elevated serum alkaline phosphatase activity (ALP) in Opg-/- mice. Interestingly, more than 50% of 2-month-old Opg-/- mice manifest medial calcification of aorta with comparable serum concentrations of calcium and phosphorus to wt mice. In conclusion, Opg-/- mice have a high-bone-rurnover type osteoporosis. The aortic calcification in Opg-/- mice is not due to abnormality of calcium and phosphorus metabolism. The mechanism underlying aortic calcification in Opg-/- mice needs to be further investigated.

Generation of Adiponectin Gene Knock-out and LacZ Gene Knock-in Mouse Model / 生物化学与生物物理进展

Adiponectin is an adipocyte-derived secretory protein. It was found to be associated with insulin resistance, inflammation and arteriosclerosis. To further study the biological function and expression of adiponection in vivo, adipoenctin gene knock-out and LacZ gene knock-in mouse model was constructed. Gene targeting strategy was designed to replace part of exon 2 and exon 3 of adiponectin gene with full length LacZ gene in frame with remaining upstream ATG and signal peptide sequence of exon 2. The targeting vector (Adipo-LacZ-XpPNT) was constructed and verified by restriction enzyme digestion and sequencing. CJ7 ES cells were transfected with targeting vector linearized by NotⅠ digestion, selected in the medium containing both G418 and ganciclovoir. Resistant clones were screened by PCR and further confirmed by Southern blot for correct homologous recombinants. Chimera mice were obtained by routing microinjection of homologous recombined ES cells into blastocysts. After mating, mice heterozygous and further homozygous for adiponectin knockout and LacZ gene knock-in were established. Expression of both endogenous adiponectin and exogenous LacZ gene in mouse tissues and sera were detected by RT-PCR, Northern-blot, Western blot and ELISA. The results show that adiponectin was disrupted at both mRNA and protein levels. LacZ gene is expressed exclusively in adipose tissue of mutant mice. Its expression profile is identical to endogenous adiponection. Unexpectedly, LacZ activity could not be detected in both adipose tissue and serum although LacZ protein can be detected in adipose tissue but not in serum of mutant mice. In conclusion, mice homozygous for adiponectin knockout and LacZ gene knock-in have been successfully constructed. Mutant mice display LacZ expression profile identical to endogenous adiponectin albeit neither LacZ activity nor protein can be detected in serum of mutant mice.

Construction and tumorigenic study on a novel fusion gene AML1-MTG16 / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To test whether splicing overlapping extension(SOE) method can be a tool for obtaining rare fusion gene's transcripts and to study the tumorigenic capacity of a novel fusion gene AML1-MTG16.</p><p><b>METHODS</b>SOE method was used to obtain AML1- MTG16 fusion gene's transcripts. MTG16, AML1-MTG16 and AML1-MTG16 without III,VI conserved domains of MTG16 segment were inserted into pEGFP- C1,pDsRed-N1 vector respectively,then transfected NIH3T3 cell line by lipofection. Forty-eight hours later, the transfected cells were examined by laser-scanning confocal microscopy. Stable transfected cells were obtained by G418 500ug/ul selection for one month. Growth curve, soft agar colonies formation tumorigenesis in nude mice were done to compare the difference between stable transfected cells.</p><p><b>RESULTS</b>Recombined AML1-MTG16 by SOE contained its CDS. NIH3T3 expressing AML1-MTG16 had a faster proliferation in medium, colony growth in soft agar. AML1-MTG16 expression cells also induced tumors formation following injection into nude mouse. MTG16,AML1-MTG16 and AML1-MTG16 without III,VI conserved domains of MTG16 were colocalized in the nucleus of cotransfected NIH3T3 cells under the examination of laser-scanning confocal microscope.</p><p><b>CONCLUSION</b>SOE is an effective method to get rare fusion gene's transcripts. AML1-MTG16 plays an important role in leukemogenesis. MTG16 may also have a carcinogenic property within the AML1-MTG16 fusion gene. Carcinogenic property of AML1-MTG16 is restricted to its localization in the nuclear matrix. N terminal of MTG16 may play an important part in the carcinogenic activity of AML1-MTG16.</p>

THE RELATIONSHIP BETWEEN TRAb AND THYROID STATUS, REMISSION OR RELAPSE OF GRAVES' DISEASE / 中华内分泌代谢杂志

TSH receptor antibodies (TRAb) were measured using radioreceptor assay in 305 patients with Graves' disease. The results of this study showed that the positive rate (94.7%) and inhibition tate (47.1?2.2 %, sx) of TRAb in hyperthyroid group were much higher than those in euthyroid group. TRAb activity was detectable in 96.9% of 32 patients with relapsed Graves' disease and the inhibition value was 50.1? 26.2%. Both values were significantly higher than those in patients who were in remission. 38 euthyroid patients were followed-up for 8-30 months after stopping the antithyroid drug. The results showed that 12 out of 16 patients who were TRAb positive relapsed, while 22 patients who were negative for TRAb remained in remission except 5 patients. The TRAb activity was higher after relapse than before (P

A RADIORECEPTOR ASSAY FOR HUMAN TSH RECEPTOR ANTIBODIES AND ITS CLINICAL APPLICATIONS / 中华内分泌代谢杂志

A radioreceptor assay for TRAb was described in which soluble TSH receptors obtained by solubilizing porcine thyroid membranes using 0.5% Triton X-100 and 125I-bTSH prepared by iodogen method and receptor-purification were used. The binding of 125I-bTSH to TSH receptors was inhibited by unlabeled bTSH and sera of patients with uncon-trolled Graves' disease (GD) in a dose-dependent manner. The assay was sensitive, specific, rapid and reproducible with interassay CV of 12.6% and 3.1% at mean inhibition values of 15.4% and 90.6% .respectively, being suitable for a clioical routine test. Of 133 patients with uncontrolled GD, TRAb was positive in 94.7%, while the absence of detectable TRAb was found in 6 patients with hyperthyroidism but not due to GD. This indicated TRAb measurement has important value in etiology and diagnosis of hyperthyroidism duc to GD.