Ahigh-resolutionmethodforhumanleukocyteantigen-B(HLA-B)genotypingwasestablished based on the optimized polymerase chain reaction, cloning and sequencing technology. The exon2 and exon3 of HLA-B gene were amplified with primers based on the HLA-B gene sequence. These produced heterozygous alleles were effectively cloned into plasmid DNA based on the principle of plasmid incompatibility, and were followed by bacterial culture. Then Sanger sequencing was carried out and after analyzing the result by software ClustalX2 and IMTG/HLA database comparison, the HLA-B genotype of the samples was achieved. Seven clinical samples were detected, and the results were consistent with those of PCR-SBT genotyping method. The method was cost-effective, high-resolution and it did not require technical software. The use of universal primers simplified the cumbersome design and optimization process of specific primers.

ObjectiveTo investigate the distribution and expression of y-secretase subunit (APH-1)in the central nervous system (CNS) of APP/PS1 double transgenic Alzheimer's disease (AD) adult mouse model,and to detect the expression difference of APH-1 in developmental brain between AD model mouse and wild-type littermates in order to further clarify the relationship between APH-1 and AD. MethodsOffspring bred by APP/PS1 double transgenic AD mice were genotyped.Immunohistochemical staining was used to detect APH-1 distribution and expression in the CNS of adult APP/PS1 double transgenic AD mouse model,in the brain of AD model mouse and its wild-type littermates on postnatal day 1,7,21 and 120.Results APH-1 was widely expressed in almost all regions of the CNS,especially in the cerebral cortex,hippocampus,olfactory bulb,hypothalamus,ventral striatum,caudate putamen,raphe magnus nucleus,cerebellum,brainstem and spinal cord of the adult APP/PS1 double transgenic mice.APH-1 expression was higher in the cortex of both AD and wild type mouse on postnatal day 1 than on postnatal day 7 and 21 with increased level of APH-1 protein in adult mouse brain.APH-1 expression in the brain of AD mice was higher than in its wild type littermates at any stage(P＜0.05).Conclusions Distribution of APH-1 is ubiquitous and region-dependent in the CNS.The different distribution and expression between APP/PS1 double transgenic mouse model and its wild type littermate indicate that APH-1 may be related to AD.

Objective To explore the relationship between serum 25-hydroxy vitamin D level and prostatic hyperplasia in elderly men.Methods Totally 95 male patients aged over 60 years were included.Blood pressure,body weight,body height,body mass index (BMI) were measured and calculated.Venous blood samples were obtained to determine fasting serum levels of 25-hydroxy vitamin D3 and blood glucose (FBG),total cholesterol (TC),triglycerides (TG),low-density lipoprotein cholesterol (LDL-C),high-density lipoprotein cholesterol (HDL-C),calcium and prostate specific antigen (PSA),total prostate volume(PV) and annual prostate growth rate were measured and calculated by ultrasound.Results The serum 25 (OH) D3 levels were varied from 12.1 nmol/L to 83.9 nmol/L,with an average of (35.5±15.2) nmol/L in elderly male patients.PV growth rate were significantly lower in elderly men with 25 (OH) D3 ＞ 50 nmol/L than in elderly men with 25 (OH) D3≤50nmol/L[(31.5± 6.0) mlvs.(39.9 ± 14.5) ml,(0.4± 0.2) ml/yvs.(0.5 ± 0.4) ml/y,P＜0.001 or 0.01].PV was negatively correlated with serum 25-hydroxy vitamin D3 level (r=-0.207,P＜0.05),and positively correlated with BMI and PSA (r=0.297,0.958,P＜0.05 or and P＜0.001).While annual prostate growth rate was positively correlated with BMI and PSA (r=0.316,0.464,P＜0.01 or ＜0.001),and positively correlated with serum 25-hydroxy vitamin D3 level,but the difference was not statistically significant (P＞0.05).Conclusions Low serum 25-hydroxy vitamin D3 level may play a role in the pathogenesis of prostatic hyperplasia.

OBJECTIVE To investigate the curative effect and mechanism of allo-bone marrow mes-enchymal stem cell(BM-MSC)infusion in the experimental autoimmune thyroiditis(EAT)mouse model. METHODS An EAT mouse model was established in C57BL/ 6 mice using porcine thyroglobulin(PTg) and Freund adjuvant,while BM-MSCs were injected into the EAT mice of BM-MSC treated group,3×105 per mouse on the 0,7th,14th and 21st day. On the 28th day,all the mice were sacrificed,and thyroid tissue was isolated,embedded in paraffin and stained with HE staining for histological examination. Serum was collected to assess the level of thyroglobulin antibodies( TgAb ) ,thyroid microsomal autoantibodies( TmAb),antithyroid peroxidase antibodies( TPOAb),3,5,3 ',5 '-tetraiodothyronine (TT4),3,5,3 '-triiodothyronine( TT3),interleukin-10( lL-10) and interferon-γ( lFN-γ ). RESULTS① Thyroid tissue in model group showed inflammatory response and infiltration. The level of TmAb, TgAb and TPOAb was significantly increased compared with normal control group,but the level of TT4 was decreased while there was no change in the level of TT3,suggesting that an EAT mouse model was established. ② The thyroid in model group and BM-MSC treated group showed inflammatory response and inflammatory cell infiltration,but the response in BM-MSC treated group was weaker than in model group. ③ Compared with model group,the level of TgAb,TmAb,TPOAb and lFN-γ was decreased obvi-ously(P﹤0.05),the level of TT4 and lL-10 was increased significantly(P﹤0.05),but the level of TT3 changed little in BM-MSC treated group. CONCLUSION BM-MSCs may partly restore the immunologi-cal homeostasis state. The mechanism may be related to its modulation of immune balance of Th1/ Th2.

Pyrosequencing is one of the important genetic polymorphism detection methods currently, but the complicated pretreatment procedure limits its application in clinical test. To simplify the whole process of pyrosequencing, on the basis of the linear_after_the_exponential_polymerase chain reaction ( LATE_PCR) , we improved the primer design method of LATE_PCR, increased the length and the concentration of the excess primer, applied direct amplification technology with whole blood, and established a whole blood_imLATE_PCR method based on common rTaq polymerase and “HpH Buffer” ( High pH buffer ) . The amplification system was optimized, and the influences of blood anticoagulant and the amount of whole blood template were investigated. The single stranded template for the pyrosequencing was obtained by PCR amplification using a single tube in one_step process, and the alcohol dehydrogenase gene polymorphisms of 24 clinical blood samples were then detected successfully. The results could be used to guide clinical individualized medication. The genotypes of ADH1B locus of 24 samples were 6 cases of AA homozygote, 14 cases of AG heterozygote, and 4 cases of GG homozygote. The genotypes of ADH1C were 20 cases of GG homozygote, 4 cases of AG heterozygote, and no cases of AA homozygote.

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.

Objective To evaluate the relationship between insulin resistance and benign prostatic hyperplasia (BPH).Methods Totally 150 male patient from the Department of Geriatrics in Peking University Hospital were included in this study.Blood pressure,body weight,body height,body mass index (BMI) were measured and calculated.Biochemical analyses including serum fasting levels of insulin(FINS),glucose,total cholesterol,triglycerides,low-density lipoprotein cholesterol,high density lipoprotein cholesterol,and prostate-specific antigen (PSA) were performed.Total prostate volume (PV) were measured by ultrasound.Results PV and annual prostate growth rate were more increased in insulin resistance group(40 cases) compared with insulin sensitivity group(110 cases) (t=2.91,3.71 respectively,both P＜0.01).Along with the levels of FINS,HOMA-IR and PSA were increased,the prostate volume was enhanced (t=-3.02,-2.88,-2.84 respectively;all P ＜0.05).PV was positively correlated with insulin resistance,serum fasting insulin and PSA (r=0.16,0.16,0.35;all P＜0.05),while annual prostate growth rate was positively related with insulin resistance,serum fasting insulin,PSA and BMI (r =0.22,0.21,0.24,0.19 ; all P ＜ 0.05).Conclusions Insulin resistance and fasting insulin plays roles in the pathogenesis of prostatic hyperplasia.

BACKGROUND: As a primary clinical predictor of fracture risk, bone mineral density (BMD) is partly genetically determined. Osteoprotegerin (OPG) is one important candidate gene in the pathogenesis of osteoporosis. OBJECTIVE: To investigate the association between T245G polymorphisms in the OPG gene and BMD. METHODS: A total of 281 elderly men and postmenopausal women, 182 males and 99 females, who received routine examinations at Peking University People's Hospital between September 2008 and April 2010 were included in this study. T245G polymorphisms in the OPG gene was detected by polymerase chain reaction-restriction fragment length polymorphism together with DNA sequencing. The BMD of the lumbar spine, Ward's triangle, and forearrm was determined by dual energy X-ray absorptiometry. Clinical variables and biochemical measurements were collected simultaneously. The association between T245G polymorphisms and each detection index was analyzed using analysis of variance. RESULTS AND CONCLUSION: The distribution of T245G genotype (alleles T, G) had no difference in elderly men or postmenopausal women (P > 0.05). The GG genotype and TG genotype had higher lumbar spine BMD and TT genotype had lower lumbar spine BMD (P < 0.05). There was no difference in BMD of the Ward's triangle or forearm among different genotypes (P > 0.05). Association between T245G polymorphism and BMD was not found in postmenopausal women. These findings indicate that OPG gene is related to lumbar spine BMD in elderly men.

Objective The invasion and metastasis of colon cancer often leads to treatment failure and mortality in patients . Our research is to investigate the influence of FoxM 1 to malignant human colon cancer line . Methods In two human colon cancer lines, the protein and mRNA expression levels of FoxM 1 were analyzed with the application of RT-PCR and Western blot , from which high-expressed HT-29 and low-expressed HCT-116 were determined.The expression of FoxM1 was down-regulated by RNA interfering in HT-29 and up-regulated by constructing overexpression transgenic line in HCT-116.The proliferation of the above cells was assayed by healing method;while the metastasis and invasion ability were examined by Transwell chamber assay . Results Two colon cancer lines were selected with high-expression or low-expression of FoxM1 separately named HT-29 and HCT-116.Application of PEX-2-FoxM1 raised after HCT-116 cells express FoxM1, cell scratches in HCT-116 experimetal group ([70.92 ±1.48]%) compared with HCT-116 control group([16.92 ±4.05]%)and HCT-116 blank control group([16.66 ±2.63]%) will markedly enhance its capabil-ity of healing (P<0.05), Transwell Chambers in membrane cells in HCT-116 experimetal group (186.0 ±6.8) compared with HCT-116 control group(42.0 ±2.0) and HCT-116 blank control grou (37.0 ± 2.2)was increased (P<0.05).On the other hand, the applied pG-PH-shFoxM1 can reduce FoxM1 expression in HT-29 cell, cell scrat-ches healing ability in HT-29 experimetal group ( [ 10 .37 ± 3.86]%) compared with HT-29 control group([34.63 ±2.35]%)and HT-29 blank control group([67.36 ±2.61]%) decreased significantly (P<0.05), Transwell Chambers in membrane cells in HT-29 experimetal group (53.0 ±1.8)compared with HT-29 control group(95.0 ±2.2)and HT-29 blank control grou(118.0 ±4.0) was also reduced (P<0.05). Conclusion The expression of FoxM1 is in close relation to the invasion and metastasis of CRC .The fact that the siRNA interfering FoxM1 could effectively inhibit the proliferation, metastasis and invasion, suggesting FoxM1 could po-tentially be a new molecular target for inhibiting the proliferation of human colon cancer line .

Objective To investigate the role of epigallocatechin gallate ( EGCG) in reversing the CpG island methylation of Rad23b and Ddit3 gene promoter and its mRNA expression induced by 0?5 Gy X-rays. Methods Thirty BALB/c male mice were randomly divided into 6 groups: control group, irradiation group, low/high dose of EGCG group, low/high dose of EGCG with irradiation group. For the irradiation group, mice were fractionally exposed with 6 MV X-rays for 10 d (0?05 Gy/d × 10 d). 2 hours after the final irradiation, all mice were killed and such tissues as blood, kidney, liver, spleen, brain, and lung were collected. Methylation and expression levels of Rad23b and Ddit3 were measured by bisulfate sequencing primers ( BSP) and Real-time PCR, respectively. Results Compare to the control group, Rad23b was hypermethylated in PBMC, liver, spleen, brain and lung (t= -20?19, -14?80, -12?05,-28?42, -12?58, P<0?05) in the irradiation group. Meanwhile, its mRNA expression level was down-regulated in PBMC, liver, brain and lung (t=25?25, 17?43, 11?53, 22?85, P<0?05). Similarly, a significant hypermethylation change of Ddit3 was observed in PBMC, liver and lung after irradiation ( t=-52?89, -20?31, -3?85, P<0?05) so that the mRNA expression of Ddit3 decreased in PBMC and liver ( t = 11?89, 16?52, P < 0?05 ). Compared to the irradiation group, EGCG with different concentrations of 10, 20 mg/kg significantly reduced the methylation level of Rad23b and Ddit3 ( t =-13?39-7?99, P<0?05), and induced re-expression of mRNA (t= -34?02 - -2?89, P<0?05). This change was more notable in the irradiation group with the high dose of EGCG. Conclusions As a natural drug, EGCG may play an important role in affecting DNA methylation and hence protects DNA from radiation damage.