Mouse Models as a Tool for Understanding Progression in Braf(V600E)-Driven Thyroid Cancers

The development of next generation sequencing (NGS) has led to marked advancement of our understanding of genetic events mediating the initiation and progression of thyroid cancers. The NGS studies have confirmed the previously reported high frequency of mutually-exclusive oncogenic alterations affecting BRAF and RAS proto-oncogenes in all stages of thyroid cancer. Initially identified by traditional sequencing approaches, the NGS studies also confirmed the acquisition of alterations that inactivate tumor protein p53 (TP53) and activate phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) in advanced thyroid cancers. Novel alterations, such as those in telomerase reverse transcriptase (TERT) promoter and mating-type switching/sucrose non-fermenting (SWI/SNF) complex, are also likely to promote progression of the BRAF(V600E)-driven thyroid cancers. A number of genetically engineered mouse models (GEMM) of BRAF(V600E)-driven thyroid cancer have been developed to investigate thyroid tumorigenesis mediated by oncogenic BRAF and to explore the role of genetic alterations identified in the genomic analyses of advanced thyroid cancer to promote tumor progression. This review will discuss the various GEMMs that have been developed to investigate oncogenic BRAF(V600E)-driven thyroid cancers.

Superoxide overproduction and kidney fibrosis: a new animal model / Produção aumentada de superóxido e fibrose renal: novo modelo animal

Objective To establish whether the mutation in the Immp2L gene induces renal fibrosis and whether aging exacerbates renal morphology in mice. Methods Female mutant mice with mutation in the inner mitochondrial membrane peptidase 2-like protein at 3 and 18 months of age were used. Renal fibrosis was analyzed using classic fibrosis score, Masson’s trichrome staining, and analysis of profibrotic markers using real time polymerase chain reaction (superoxide dismutase 1, metalloproteinase-9, erythropoietin, transforming growth factor beta), and immunostaining (fibroblasts and Type IV collagen). Oxidative stress markers were determined by immunohistochemistry. The number of renal apoptotic cells was determined. Renal function was estimated by serum creatinine. Results Young mutant mice had significantly more glomerulosclerosis than age-matched mice (p=0.034). Mutant mice had more tubular casts (p=0.025), collagen deposition (p=0.019), and collagen type IV expression (p<0.001). Superoxide dismutase 1 expression was significantly higher in young mutants (p=0.038). Old mutants exhibited significantly higher expression of the fibroblast marker and macrophage marker (p=0.007 and p=0.012, respectively). The real time polymerase chain reaction of metalloproteinase-9 and erythropoietin were enhanced 2.5- and 6-fold, respectively, in old mutants. Serum creatinine was significantly higher in old mutants (p<0.001). Conclusion This mutation altered renal architecture by increasing the deposition of extracellular matrix, oxidative stress, and inflammation, suggesting a protective role of Immp2L against renal fibrosis. .

Objetivo Estabelecer se a mutação no gene Immp2L induz à fibrose renal e se o envelhecimento exacerba a morfologia renal em camundongos. Métodos Foram usadas fêmeas de camundongos mutantes para proteína semelhante à peptidase 2 da camada interna da mitocôndria, com 3 e 18 meses de idade. Para analisar a fibrose renal, foram usados o escore clássico de fibrose, a coloração com tricrômio de Masson, e a análise de marcadores profibróticos, por meio da reação em cadeia de polimerase em tempo real (superóxido dismutase 1, metalonoproteinase-9, eritropoietina e fator transformador de crescimento beta), e a imunocoloração (fibroblastos e colágeno IV). Marcadores de estresse oxidativo foram determinados por imuno-histoquímica. O número de células apoptóticas renais foi analisado. A função renal foi estimada por creatinina sérica. Resultados Camundongos mutantes jovens apresentaram glomeruloesclerose em quantidade significativamente maior que animais da mesma idade (p=0,034). Os mutantes mostraram maior formação de cilindros tubulares (p=0,025), deposição de colágeno (p=0,019) e maior expressão de colágeno do tipo IV (p<0,001). A expressão de superóxido dismutase 1 foi maior em mutantes jovens (p=0,038). Mutantes idosas exibiram maior expressão dos marcadores de fibroblastos e macrófagos (p=0,007 e p=0,012, respectivamente). As reações da cadeia de polimerase em tempo real da metalanoproteinase-9 e da eritropoietina estavam aumentadas em 2,5 e 6 vezes, respectivamente, em mutantes idosas. A creatinina sérica foi significantemente maior em animais idosos mutantes (p<0,001). Conclusão Essa mutação alterou a arquitetura renal pelo aumento da deposição de matriz extracelular, estresse oxidativo e inflamação, sugerindo papel de proteção de Immp2L contra a fibrose renal. .

Effect of hepatopoietin Cn on liver stem cells / 军事医学

Objective To investigate the effect of hepatopoietin Cn(HPPCn) on liver stem cells.Methods In this study, WB-F344 cell line was used, and MTT and flow cytometry assay were conducted to determine cell proliferation and apoptosis.Transwell assay was used to test the migration of WB-F344 cells.A 2AAF-partial hepatectomy(PH) mouse model was used to observe the effect of HPPCn on liver stem cell proliferation in vivo.Results HPPCn enhanced WB-F344 cell proliferation and migration and activated the SphK1, Erk and Stat3 signal pathways.The analysis of the 2AAF-PH mouse model showed that oval cells in the experimental group far outnumbered those in control and the regeneration of the liver was improved post PH.Conclusion HPPCn can increase the liver stem cell proliferation and survival while promoting the regenenation of the liver by augmenting oval cell proliferation.

The impact of celastrol on cognitive function and expressions of Aβ40 and Aβ42 in hippocampus in APPswe/PS1dE9 double transgenic mouse after partial hepatolobectomy / 中国医师杂志

Objective To investigate the effect of celastrol on space learning capability and expressions of beta-amyloid (Aβ) 40 and Aβ42 in hippocampus in APPswe/PS1dE9 double transgenic mouse after partial hepatolobectomy.Methods The 3-month-old APPswe/PS1dE9 double transgenic mice (n =96) were randomly divided into three groups according to the random number table method.Surgery group (group S, partial hepatolobectomy;n =32), celastrol group (group C, injections of dimethyl sulphoxide/DMSO and celastrol for 3 days before undergoing partial hepatectomy, on the surgery day, and for a further 4 days after surgery;n =32), and DMSO group (group D, injections of DMSO for 3 days before undergoing partial hepatectomy, on the surgery day, and for a further 4 days after surgery;n =32).Eight mice were selected randomly in each group and were Morris-water maze trained for continuous 5 days.Theirs learning and memory abilities were evaluated at 1,3, 7 and 14 d after surgery, respectively.Hippocampus was collected and the changes of β40 and Aβ42 were measured by enzyme-linked immunosorbent assay (ELISA) at the time set in advance in each group.Results The average escape latency of group C was significantly shorter than groups S and D at 3, 7 and 14 d after partial hepatectomy (P ＜ 0.05).Times of passing through the platform groups S and D were significantly less than group C (P ＜ 0.05).The expressions of Aβ40/Aβ42 in group C were lower than group S and group D at 1, 3, 7 and 14 d after partial hepatectomy (P ＜ 0.05).Conclusions Through decreasing the expressions of Aβ40 and Aβ42 in hippocampus,celastrol improves the space learning capability in APPswe/PS1dE9, the double transgenic mouse after partial hepatolobectomy.

The Specificity and Efficiency of YFP Labeled NK Cells through Vav-Cre Induced YFP Reporter System in Mice / 天津医药

Objective To explore the specificity and efficiency of YFP labeled natural killer (NK) cells through Vav-Cre induced YFP reporter system in mice. Methods ROSA26R-YFP and Vav-Cre mice were crossed, and their YFP and Cre gene double positive progeny were screened by genotyping. The specificity of YFP in hematopoietic cells from im-mune organs including lymph nodes, spleen, thymus and bone marrow were analyzed by flow cytometry. The percentages of YFP positive cells in NK cells from lymph nodes, spleen and bone marrow were also analyzed by flow cytometry. Results A total of 11 double positive mice (ROSA26R-YFP-(+/-)VavCre) were obtained in 17 mouse offspring by crossing ROSA26R-YFP mice with Vav-Cre mice. The percentages of YFP positive cells in immune organs including lymph nodes, spleen, thy-mus and bone marrow were 73.87%± 1.51%, 56.07%± 1.47%, 86.17%± 1.74%and 53.60%± 3.56%, and there were signifi-cant differences compared with the corresponding negative control cells(0.27%±0.01%, 1.33%±0.91%, 0.11%±0.01%and 0.29%± 0.03%, P0.05). The positive rates of YFP were significantly higher in NK cells in lymph nodes, spleen and bone marrow (76.94%±0.84%、81.66%±1.18%and 88.92%±0.77%) compared with those of control (P<0.01). Conclusion YFP marked NK cells through Vav-Cre induced YFP reporter system in mice have high specificity and efficiency.

The Effects ofβ-Sheet Breaker Peptide H102 on ERK Signal Transduction Pathway in Brain of PAP Double Transgenic Mice / 天津医药

Objective To investigate the activation of β-sheet breaker peptide H102 on ERK signal transduction pathway in brain of PAP double transgenic mice. Methods PAP double transgenic mice were randomly divided into model group and H102 treatment group (n=10 for each group). A group of C57BL/6J mice with the same genetic background was served as controls. H102 (5.8 mg/kg) 5 μL was infused by intranasal administration to mice in H102 treatment group, and equal volume of blank solution of H102 (chitosan, BSA) was given to mice in control group and model group. The ability of spatial reference memory was tested by Morris water maze after 30 days of treatment. Then immunohistochemistry tests and Western blot technique were used to detect the content of RAS, P-MEK and P-ERK proteins in mouse brain. Results (1) The ability of learning and memory was significantly lower in model group than that of control group. The ability of learning and memory was significantly improved in treatment group than that in model group (P<0.05). (2) The contents of RAS, P-MEK and P-ERK in mouse brain were significantly lower in model group than those of control group, and these protein ex-pressions were significantly increased in treatment group than those in model group (P<0.01). Conclusion β-sheet break-er peptide H102 can activate ERK signal transduction pathway in brain of PAP double transgenic mice, increase PAS, P-MEK and P-ERK levels in nerve cells, and improve the ability of learning and memory in PAP mice.

Effects of hepatolobectomy on space learning capability and hippocampus Drebrin and PSD95 expressions in APP/PS1 transgenic mouse model of Alzheimer's disease / 中华老年医学杂志

Objective To study the influences of hepatolobectomy on space learning capability and the expressions of Drebrin and PSD95 in the hippocampus in APP/PS1 transgenic mouse model of Alzheimer's disease.Methods A total of fifty-four 3-month-old APP/PS1 transgenic mouse were randomly divided into 3 groups:3-month-old control group,sham surgery group,hepatolobectomy group,3-month-old littermates control group and 6-month-old control group.Morris water maze test was used to observe space learning capability on the 1st,3rd,7th,14th day after surgery,meanwhile the expressions of Drebrin and PSD95 in hippocampus were measured by Western blotting.Results Compared with the sham surgery group,the results of the Morris water maze test were decreased in hepatolobectomy group at day 3,7,14 after surgery [(62.9±6.9) s vs.(35.7±12.2) s,(66.3± 9.5) s vs.(39.3±8.3) s,(67.1±7.5) s vs.(32.6±14.1) s],and 6-month-old control group [(75.9±12.1) s] (all P＜0.05).The escape latency were (62.9±6.9)s,(66.3±9.5)s,(67.1± 7.5)s and (75.9±12.1)s,the probe trials were (2.1±0.7) times,(1.83±1.5) times,(2.5±1.9) times and (1.8±0.8) times respectively in hepatolobectomy group at day 3,7,14 after surgery and 6-month old control group.No significant differences in the results of the Morris water maze test were found among 3-month-old control group,3-month-old littermates control group,and sham surgery group.Compared with 3-month-old control group and sham surgery group atday 1,3,7,14 after surgery the expressions of Drebrin were decreased in 6-month-old control group and the hepatolobectomy group at the same time points.Meanwhile,the expression of Drebrin in hepatolobectomy group was increasedat day 14 versus day 7 after surgery.Compared with 3 month-old control group,the expression of Drebrin was increased at day 7 after sham surgery.Compared with 3-month-old control group and sham surgery group at day 3,7 and 14,the expressions of PSD95 were decreased in hepatolobectomy group at the same time points.Compared with 3-month-old control group,the expression of PSD95 was increased in sham surgery group at 7th day (P＜0.05).Between 3 month-old control group and 3-month-old littermates control group,the expressions of Drebrin and PSD95 had no significant differences.Concltsions Hepatolobectomy can impair the capabilities of space learning and memory in 3-month-old APP/PS1 transgenic mice,which may be associated with the decreased expressions of Drebrin and PSD95 in hippocampus.

The study on pathological changes of tau protein in the APP/tau/PS1 triple transgenic Alzheimer's disease mice / 中华老年医学杂志

Objective To identify the genotype of the APP/tau/PS1 triple transgenic Alzheimer's disease (AD) mice,and investigate the pathological changes of tau protein in the pathogenic process.Methods Using specific primers of PS1,APP,tau gene,the genotypes of the triple transgenic AD mice were identified.Expression of tau protein in hippocampal tissue of mouse model aged 2,4,8 month was detected by immunohistochemistry.The expression of tau and its hyperphosphorylation in different sites in the hippocampal tissue and different month old mice was detected by Western blotting.Results PCR amplification fragment of 960 bp,530 bp and 400 bp of transgenic mouse genome were the expected size of APP,PS1,tau,respectively.Expression of tau in hippocampal CA3 region was increased obviously in the 8 month old mice.Compared with the normal wild-type mice,the expressions of tau and phosphorylation of pS262,pS404 and pS202 were increased significantly in hippocampus tissue of the transgenic mice (P＜0.01).Expression of tau were significantly higher in 8-and 12-monthsold mice than in 2 months-old mice (P ＜ 0.01).Phosphorylation level of pS404 and Ps202 was significantly increased since 2-months-old in transgenic mouse compared to the wild type mouse (P＜0.01),and in 8-monthold mice,there was also a significant increase as compared to that in 2 month-old mice (P＜0.01).As to the phosphorylation level of pSs262,the significant increase did not appear until 12 months old in transgenic mouse as compared to the wild type mouse (P＜0.01).Conclusions The triple transgenic mice can stably express the APP/tau/PS1 gene.The transgenic animals can be a useful model with the pathological features of tau of AD.The phosphorylation level of tau in different site increases in different time,which will provide useful research reference in Alzheimer's disease pathology and medication research.

Effect of catalpol on senile plaques and spatial learning and memory ability in amyloid-β protein precursor/presenilin 1 double transgenic mice / 中华神经科杂志

Objective To investigate whether catalpol affects senile plaque formation and spatial learning and memory ability in the amyloid-β protein precursor/presenilin 1 (APP/PSI) double transgenic mice.Methods Three month-old APP/PS1 double transgenic mice were randomly divided into catalpoltreated and saline-treated groups (n =10),with C57 mice of the same age and genetic background as normal control group (n =10).The catalpol (in a dose of 5 mg · kg-1 · d-1) and the same amount of saline were peritoneally injected into Alzheimer' s disease (AD) model mice for 3 weeks.Immunohistochemical staining was performed to examine senile plaques in the brain of AD model mice,and Morris water maze was used to assess the spatial learning and memory abilities of the mice.Results Compared with the saline-treated AD model mice (6.0 ±0.6),the number of senile plaques of catalpol treated AD mice significantly decreased (2.3± 0.7; t =3.500,P =0.025); Mice in each groups had similar latency and path length to reach platform in visible platform test; In hidden platform test,catalpol-treated mice had a significant lesser latency and path length compared with saline-treated mice,furthermore,catalpol-treated mice had much more platform-crossing times (6.4 ± 0.8) than saline-treated mice (2.9 ± 0.4 ; t =5.592,P =0.001).Conclusion Catalpol can significantly decrease the senile plaque formation and improve the spatial learning and memory abilities of APP/PS1 double transgenic mice.

Effect of transplantation of mouse marrow stromal cells through injection into cerebrospinal fluid on Cu/Zn superoxide dismutase 1 transgenic mice / 中华神经科杂志

Objective To investigate the effect of transplantation of mouse marrow stromal cells (mMSCs) on Cu/Zn superoxide dismutase 1 (SOD1) transgenic mice through cerebrospinal fluid (CSF) injection.Methods We delivered 5 × 105 mMSCs into SOD1 transgenic mice by single injection to CSF at the age of 8 weeks or by multiple injections at the age of 8,10 and 12 weeks.Neurologic phenotype observation,weight measurement,hanging wire test and motor neuron counts were used to assess disease progression of SOD1 mice.Results Single transplantation of mMSCs didn' t show beneficial effect on SOD1 mice.Compared with vehicle-treated mice,SOD1 transgenic mice with multiple transplantations of mMSCs demonstrated attenuated weight loss,improved motor performance,decreased motor neuron loss,and importantly,prolonged survival by 17 days ((157 ± 15) d vs (140 ± 11) d; x2 =7.56,P ＜ 0.01).Conclusion Intrathecal injection could be an effective route of cell transplantation,and multiple transplantations of mMSCs are needed to achieve the therapeutic effect on SOD1 mice.

Establishment of transgenic mouse models expressing human HSP22 protein / 中华老年医学杂志

Objective To establish transgenic mouse models expressing human HSP22 protein.Methods pCAGGS-HA-Wt HSP22 transgenic expressing vector carrying human HSP22 gene was constructed by gene recombination technology.The linearized DNA was got by SalI、Hind Ⅲ and BsaⅪ digestion of PCAGGS-HA-Wt HSP22,purified and microinjected into fertilized eggs from C57BL mice.The tail DNA of pups was tested by PCR and DNA sequencing.Expression of human HSP22 protein was detected by western blot with anti-HA tag monoclonal antibody.Results 4 transgenic founder mice (Tg646,Tg648,Tg649,Tg661) carrying human HSP22 gene were identified by PCR and DNA sequencing.The human HSP22 protein was expressed in the lines Tg646,Tg648 and Tg649 founder mice,but was not expressed in the line Tg661 founder mouse.Conclusions The mouse models expressing human HSP22 protein are established successfully and provide the foundation for HSP22 gene research in vivo.

The effects of suberoylanilide hydroxamic acid on cognitive performance and cerebral tau phosphorylation in Alzheimer's disease transgenic mice / 中华神经科杂志

Objective To observe the effects of histone deacetylases inhibitor (HDACi) on cognitive performance and cerebral tau phosphorylation in transgenic mice coexpressed five familial Alzheimer' s disease mutations (5XFAD).Method The total 12 5XFAD-CC and 12 wild type (WT) mice were administrated with suberoylanilide hydroxamic acid ( SAHA,n =7) and vehicle ( n =5 ),respectively.The cognitive performance was assessed by Y-maze and Morris water maze.The protein levels of acetylated α-tubulin,total tau and phosphorylated tau and phosphorylated glycogen synthase kinase-3β (GSK3β) were determined by Western blotting.Results SAHA ameliorated learning and memory deficits in 5XFAD-CC mice (39.10％ ±2.25％,t =2.688,P =0.0312 for total numbers of entrance in novel arm; 26.81％ ±0.78％,t =3.271,P =0.017 for time spending in novel arm; F =5.936,P =0.045 for hidden platform;31.70％ ±4.21％,t =2.317,P =0.049 for probe trial).Administration of SAHA significantly increased acetylated α-tubulin in hippocampus of WT and 5XFAD-CC mice (26.42％ and 29.64％,respectively).Additionally,SAHA attenuated tau-pSer396,tau-pSer404 and tau-pThrThr231 in hippocampus of 5XFAD-CC mice (24.22％,48.98％ and 26.95％,respectively). Moreover,hippocampal phosphorylated GSK3β was markedly reduced in SAHA-treated 5XFAD-CC mice (31.29％). Conclusion SAHA may improve cognitive performance in 5XFAD-CC mice, which is associated with its significant effects on the phosphorylation of tau and GSK3β.

1H-MR spectroscopy in evaluating the effect of neural stem cell transplantation on Alzheimer's disease in an APP-PS1 transgenic mouse model / 中华放射学杂志

Objective To explore the value of 1H-MRS on the evaluation of Alzheimer's disease (AD) with neural stem cells (NSCs) transplantation in an APP-PS1 double transgenic (tg) AD mouse model.Methods NSCs from C57BL/6 mice were cultured and amplified.APP-PS1 tg mice (n =30) aged 12 months were used as the study group,and mild-type mice (n =15) were used as the control group.Animals in the study group were randomized into two subgroups,the AD mice in one subgroup received NSCs transplantation (NSCs group) and in another subgroup received phosphate buffer saline (PBS,PBS group)in bilateral hippocampal CA1.Animals in the control group were not treated.Using a 7.0 T high-fieldstrength MR imager,1H-MRS was performed before and 6 weeks after transplantation to measure the area under the peak of n-acetyl aspartate (NAA),glutamate (Glu),myo-inositol ( mI),choline (Cho) and creatine (Cr) in the hippocampal area,NAA/Cr,Glu/Cr,mI/Cr and Cho/Cr ratio were calculated and compared with histopathological results (including Nissl's staining and electron microscope examination).Comparisons among NSCs,PBS and control groups were conducted by one-way ANOVA.Results NSCs from C57BL/6 mice were cultured successfully. Before transplantation,the mean NAA/Cr,Glu/Cr and mI/Cr in NSCs,PBS and control groups were 0.89 ± 0.05,0.88 ± 0.04 and 1.15 ± 0.05,0.40 ± 0.03,0.39 ± 0.03 and 0.45 ± 0.05,0.67 ± 0.05,0.67 ± 0.05 and 0.52 ± 0.04,respectively,and differences were statistically significant (F =148.918,7.529,59.468,P ＜ 0.01 ). There were no significant differences in NAA/Cr,mI/Cr and Glu/Cr ratios between NSCs and PBS groups before transplantation (t =0.147,0.096,0.207,P ＞ 0.05 ),but the differences were significant compared with the control group (t =0.255,0.467,0.171 and t =0.269,0.527,0.151,P ＜0.05).Six weeks after transplantation,the mean NAA/Cr,Glu/Cr and mI/Cr in three groups were 1.13 ±0.07,0.86 ±0.05 and 1.14 ±0.05,0.45 ± 0.04,0.38 ± 0.02 and 0.44 ± 0.03,0.58 ± 0.04,0.67 ± 0.04 and 0.53 ± 0.04,respectively,and differences were statistically significant ( F =112.092,23.076,44.367,P ＜ 0.01 ).NAA/Cr and Glu/Cr ratios were increased and mI/Cr was decreased in NSCs group,and the difference was significant compared with PBS group at the same time point ( t =0.271,0.071,0.089,P ＜ 0.05 ).There were no significant differences in NAA/Cr and Glu/Cr ( t =0.013,0.012,P ＞ 0.05 ),but there was a significant difference in mI/Cr between NSCs and control groups ( t =0.046,P ＜ 0.05).There were no significant differences in Cho before and after transplantation among the three groups (P ＞ 0.05 ). Nissl's staining showed that the number of neurons in the hippocampal area increased more significantly in tg mice receiving NSCs than that without receiving NSCs.Electron microscopy showed that most hippocampal NSCs in NSCs group were morphologically normal with abundant organelles,while hippocampal NSCs in PBS group were swollen with sparse synapses.Conclusion 1H-MRS is able to display intracranial metabolite changes before and after NSCs in APP-PS1 double transgenic AD mice and has an applicable value in evaluating the therapeutic effect of NSCs on AD.

Study on the immune tolerance status and blood biochemical index of hepatitis B virus transgenic mice / 中华传染病杂志

ObjectiveTo research the hepatitis B virus (HBV) replication and immune tolerance status of transgenic mice for elucidating the pathogenesis of hepatitis B and evaluating new drugs against HBV.Methods SPE grade HBsAg negative nontransgenic and transgenic mice with the same genetic background were recruited in this study.HBsAg,HBeAg and HBV DNA were detected by chemiluminescent method.Pre-S1 and HBcAg were detected by enzyme linked immunosorbont assay (ELISA).Liver pathology was examined and HBsAg expressions at different stages were determined by immunohistochemical staining.The lymphocyte proliferation of mice was detected by flow cytometry and interferon (IFN)γ-producing T lymphocytes was determined by enzyme linked immunospot (ELISPOT).The expressions of Toll-like receptor (TLR)2 and TLR9 in splenocyte suspension and splenic dendrite cells (DC) were determined by double-labeling immunofluorescence.The data were analyzed by t test and F test.ResultsHBsAg,preS1,HBeAg,HBcAg were expressed and HBV DNA was replicated in HBV transgenic mice,while anti-HBs,anti-HBc,and anti-HBe were all negative.There were no obvious pathological changes in liver tissues.HBsAg was expressed in cytoplasm and HBcAg in nucleus of hepatocytes.After stimulated with HBsAg,T lymphocyte proliferation capacity of HBV transgenic mice was (697.6±67.3) cpm,which was much lower than that of nontransgenic mice [( 1315.5 ±191.6) cpm].The number of spot forming cells of IFNγ-producing splenocytes from transgenic mice after HBsAg stimulation was 8.25 ± 1.10,which was obviously lower than that of nontransgenic mice (28.50±4.21) (F=155.967,P=0.000).The expressions of CD11c+,TLR2 and TLR9 on DC from both HBV transgenic and nontransgenic mice were not different significantly (all P＞0.05).The HBsAg expressions in liver tissues were observed in 18-day-old fetal mice and 1-day-old newborn mice.ConclusionsThe HBV transgenic mice can express HBV-related antigens,and are immune tolerant to the antigens.The innate and acquired immunity of the HBV transgenic mice are normal,which is similar to chronic asymptomatic HBV carriers of human.Therefore,HBV transgenic mouse is an ideal animal model.

Effect of human mesenchymal stem cells intracardiac transplantation on superoxide dismutase 1-G93A mice / 中华神经科杂志

Objective To study the changes of life span and pathology in superoxide dismutase 1 (SOD1)-G93A mice after intracardiac transplantation of human mesenchymal stem cells (hMSCs).Methods hMSCs were isolated from bone marrow cells obtained from healthy donors and cultured.The purity and morphology were assessed by flow cytometry (FCM).hMSCs (3×10~6) resuspended in 0.2 ml DMEM was injected into the heart of 8 week-old SOD1-G93A mice.In non-transplantion control SOD1-G93A mice, only DMEM was injected.The mice were evaluated for signs of motor deficit with 4-point scoring system previously described by Weydt et al.The age of onset and life span in mice were assessed.The pathological change including number of motor neurons was investigated by Nissl staining.Immunofluorescence staining with specific human nuclear antibody was used to confirm the transplant of hMSCs in mice.Results The onset symptoms in untreated SOD1-G93A mice appeared at (156.56±3.60) days of age and the average life span was (188.32±3.51) days.hMSCs transplantation delayed the onset of ALS type symptoms about 16 days (x~2=10.888, P=0.001) and prolonged the life span about 14 days compared to the untreated SOD1-G93A littermates((202.19±4.09) days vs (188.32±3.51) days, x~2=3.917, P=0.04).The loss of motor neurons in untreated mice was earlier and more severe than in hMSCs transplanted mice.At 20 weeks, the number of motor neurons in transplanted mice was significantly higher than those in untreated mice.Human specific nuclear antigen in brain and spinal cord was detected in transplanted SOD1-G93A mice.Conclusion hMSCs can be implanted for a long-term into central nervous system by intracardiac transplantation and the transplantation can prolong life span, and delay the onset of the disease and motor neuron loss in SOD1-G93A mice.

Iron and senile plaques deposition in transgenic mouse model of Alzheimer's disease and influence on MR T2 relaxation times / 中华神经科杂志

Objective To observe senile plaque and iron deposition in cortex and hippocampus of the Alzheimer's disease ( AD ) transgenic mice and investigate their influence on T2 relaxation time.Method All AD transgeic mice were divided into three groups: young group(2,4 months), adult group (6,8,10 months), old group (12,14,16 months), and C57BL/6J mice were as control and were scanned in order by using 4.7 T MR system.Regions of interest (ROI) corresponding to cortex, hippocampus,thalamus, striatum were manually drawn on MR images and T2 MR relaxation times of each ROI were calculated.After MR scan, these mice were decapitated and stained for iron and senile palques.The number of plaque and iron, plaque burden, iron load in cortex and hippocampus were acquired using image pro plus software.Result T2 relaxation times of each group were as following: wild type ( cortex (49.5 ± 2.1 ) ms,hippocampus (51.6 ± 1.1 ) ms ); young ( cortex ( 49.7 ± 0.5 ) ms, hippocampus ( 50.7 ± 0.7 ) ms ); adult (cortex(47.2 ±0.8) ms, hippocampus(47.7 ±0.9) ms) and old (cortex(44.6 ±0.8) ms, hippocampus (45.3 ±0.4)ms).T2 relaxation times in cortex and hippocampus of each group had statistical differences ( cortex F = 18.620, P ＜ 0.01; hippocampus F = 67.925, P ＜ 0.01 ); Compared with young group and wild type mice, T2 relaxation times in corex and hippocampus of adult group mice were decreased significantly.At the same time, T2 relaxation times in old group mice were reduced compared with adult group ( Adult vs young: cortex q =4.284, P ＜0.01, hippocampus q =7.902, P ＜0.01; adult vs wild type: cortex q =4.424, P＜0.05, hippocampus q = 11.450, P ＜0.01; old w adult: cortex q =4.812, P ＜0.01,hippocampus q = 7.034, P ＜ 0.01 ).Histochemical staining for senile plaques found that senile plaques was deposited as early as 4 month.Iron deposition in hippocampus and cortex were detected by perl-DAB as early as 6 months of age, and there was an overall increase in number and load of plaques and iron with age.A positive correlation was observed between plaque burden and iron load ( r = 0.931, P ＜ 0.01 ).At the same time, plaque burden and iron load were negatively correlated with T2 relaxation times ( plaque burden and T2 relaxation times r = - 0.884, P ＜ 0.01; iron load and T2 relaxation times r = - 0.827, P ＜ 0.01 ).Conclusion The changes of T2 relaxation time in AD transgenic mice are attributed to iron and senile plaques.MR T2 relaxation time is a sensitive marker to diagnosis for AD and screen antidementia drugs.

Tumorigenesis after Injection of Lung Cancer Cell Line (SW-900 G IV) into the Pleural Cavity of Nude Mice / 대한흉부외과학회지

BACKGROUND: Base on types of tumor, the types of expressed tumor is diverse and the difference in its expression rate is even more various. Due to such reasons an animal model is absolutely needed for a clinical research of lung cancer. The author attempted oncogenesis by cultivating a cell line of non-small cell carcinoma and then injecting it inside thoracic cavities of nude mice. The author conducted quantitative analyses of HER2/neu tumor gene - an epidermal growth factor receptor (EGFR) related to lung cancer, and TGF-beta1, which acts as a resistance to cell growth inhibition and malignant degeneration. In order to investigate achievability of the oncogenesis, histological changes and the expression of cancer gene in case of orthotopic lung cancer is necessary. MATERIAL AND METHOD: Among 20 immunity-free male BALB/c, five nude mice were selected as the control group and rest as the experimental group. Their weights ranged from 20 to 25 gm (Orient, Japan). After injection of lung cancer line (SW900 G IV) into the pleural cavity of nude mice, They were raised at aseptic room for 8 weeks. HER2/neu was quantitatively analyzed by separating serum from gathered blood via chemiluminiscent immunoassay (CLIA), and immunosandwitch method was applied to quantitatively analyze TGF-beta1 SPSS statistical program (SPSS Version 10.0, USA) was implemented for statistical analysis. Student T test was done, and cases in which p-value is less than 0.05 were considered significant. RESULT: Even after lung cancer was formed in the normal control group or after intentionally injected lung cancer cell line, no amplification of HER2/neu gene showed reaction. However, the exact quantity of TGF-beta1was 28,490+/-8,549 pg/mL, and the quantity in the group injected with lung cancer cell was 42,362+/-14,449 pg/mL, meaning 1.48 times highly significant (p<0.483). It proved that HER2/neu gene TGF-beta1had no meaningful interconnection. CONCLUSION: TGF-beta1gene expressed approximately 1.48 times amplification in comparison to the control group. The amplification of TGF-beta1meant somatic recuperation inhibition mechanism due to carcinogenesis in nude mice was definitely working. It may be implemented as a quantitative analysis that allows early detection of lung cancer in human body.

The presence of CD8+ invariant NKT cells in mice

Invariant natural killer T (iNKT) cells develop in the thymus upon recognition of CD1d expressed on developing thymocytes. Although CD4 and CD8 coreceptors are not directly involved in the interaction between CD1d and the T cell receptors (TCRs) of iNKT cells, a conspicuous lack of CD8+ iNKT cells in mice raised the question of whether CD8+ iNKT cells are excluded due to negative selection during their thymic development, or if there is no lineage commitment for the development of murine CD8+ iNKT cells. To address this question, we analyzed iNKT cell-specific TCR Valpha14+ transgenic mice, where the Valpha14 transgene forces the generation of iNKT cells. This allows detailed study of the iNKT cell repertoire. We were able to identify CD8+ iNKT cells which respond to the NKT cell-specific glycolipid ligand alpha-galactosylceramide. Unlike conventional iNKT cells, CD8+ iNKT cells produce predominantly IFN-gamma but not IL-4 upon antigen stimulation. We also confirmed the presence of CD8+ iNKT cells in wild type mice. Our results suggest that CD8+ NKT cells do exist in mice, although their population size is quite small. Their Th1-skewed phenotype might explain why the population size of this subtype needs to be controlled tightly.

Generation of transgenic mice with liver-specific expression of delta drosophila homolog-like 1 / 中华消化杂志

Objective To establish and identify transgenic mice model that specifically express delta drosophila homolog-like 1 (DLK1) in liver for the functional study of DLK1 on liver differentiation,hepatopathy,and hepatocellular carcinoma.Methods The coding sequence of DLK1 cDNA was inserted downstream of mouse albumin gene enhancer/promoter to construct a liver-specific DLK1 expression recombinant vector.The DNA fragment of transgene digested from the recombinant vector by Pine I was transfected to Hepl-6 cells to verify the expression of DLK1 in vitro.Then the transgene fragment was microinjeeted into fertilized eggs of C57×CBA F1 mice and 20 transgenic founder mice were generated.The F1 mice of DLK1 transgenie founder mice were used to identify the expression of the transgene in liver and other tissues.Results RT-PCR and cellular immunofluorescence showed DLK1 expression when the transgene fragment was transfeeted into Hep1-6.For transgenic mice,RT-PCR and immunohistochemistry showed that DLK1 was specifically expressed in adult F1 mice liver.Conclusion Successfully established the liver-specific DLK1 expression transgenic mice model.

Immunophenotyping of myeloid leukemia in transgenic mouse model / 中华检验医学杂志

Objective To analyze the immunophenotyping characteristics of myeloid leukemia in transgenic mouse.Methods According to differential antigen expression profile on various hematopoietic lineages,flow cytometric analysis of bone marrow sample was performed on 5 myeloid leukemia mice and 10 healthy BL6 mice.Cell cycle analysis was further performed to assess cell proliferation.Results Expressions of Mac-1+ Gr-1+ and c-Kit+ in bone marrow cells in transgenic leukemia mice were(72.6±6.5)% and (20.5±4.8)%,and it were significantly higher than those in normal mice[(52.8±4.8)% and(2.1±0.3)%](t=6.66,12.66,P＜0.01).And expressions of B220+,CD3+,CD41+ and Ter119+ in leukemia mice were(2.7±1.1)%,(1.2±0.3)%,(1.2±0.6)% and(2.8±1.1)%,respectively.It were significantly lower than those in normal mice[(20.2±2.1)%.(6.6±1.3)%,(4.7±1.1)% and(10.6±1.2)%](t=-17.63,-8.69,-6.30,-12.28,P＜0.01).The percentages of S phase and G2/M phase in leukemia mice were(25.7±4.2)% and(21.1±4.2)%,respectively.It were significantly increased as compared with normal mice[(11.8±2.1)% and(8.9±1.8)%](t=8.59,7.98,P＜0.01).Conclusions Immunophenotyping of myeloid leukemia in transgenic mouse was characterized by hish expression of myeloid specific marker(Mac-1 and Gr-1)and hematopoietic stem/progenitors cells specific marker(c-Kit),and by low expression of B-lymphoid specific marker(B220),T-lymphoid(CD3),megakaryocyte(CD41)and Erythroid(Ter119).