Objective To optimize the method for total DNA extraction and RAPD analysis of Nervilia fordii (NF),and to study the genetic diversity of different breeds of NF,the substitute of NF and the fake of NF at molecular level.Methods We used low-pH extraction medium with high salt to extract total DNA,and used randomly amplified polymorphic DNAs (RAPD) to select polymorphism primer from 49 random primers.Twenty-two kinds of NF samples were analyzed by RAPD,and classified by SPSS.Genetic diversity were estimated by Shonnon's index and Nei's index.Results A higher puritiy of DNA can be obtained from fresh HF than that from medical materials.We selected 19 polymorphism primers for the cluster analysis of fresh NF and dried medical material.For medical materials of NF,the distance of amplification band of small-leave breed is close to that of middle-size leave breed,but is far away from that of,big-leave breed and Nervilia plicata.For the fresh materials of NF,there breeds of Nervilia fordii can be classified into one kind,the distance of fresh NF band is far from that of Nervilia plicata,Pachyrhizus erosus and the cultured breed,and more far awary from that of Plantago asiatica and Centella asiatica.Shonnon's genetic diversity is 0.463,Nei's genetic diversity is 0.267.Intra-population genetic variation is obvious compared to iner-population genetic variation.The estimated gene flow from Gst (Nm) is 0.94.Conclusion The molecular difference in different breeds of Nervilia Fordii can be used to identify Nervilia Fordii.The genetic diversity of Nervilia fordii is mainly caused by the geography environment.

Adult ; Aminosalicylic Acid ; therapeutic use ; Female ; Humans ; Manganese Poisoning ; drug therapy

Objective To investigate the antitumor effect of tumor necrosis factor-related apoptosis-inducing ligand(TRAIL) gene transfection mediated by adenovirus into human pancreatic carcinoma cell line Panc-1,and the mechanisms involved in this effect.Methods TRAIL gene was transfected into pancreatic cancer cell line Panc-1 by an adenovirus vector(Ad-TRAIL).Level of TRAIL mRNA expression was determined using RT-PCR,and TRAIL protein synthesis was evaluated with Western blot.Cell-growth activities were determined by MTT assay.The bystander effect was observed by co-culturing the Panc-1 cells with and without the transfected TRAIL gene at different ratios.Apoptosis in pancreatic cancer cells was detected by flow cytometry.Proaspase-8 and procaspase-3 proteins were determined by Western blot.Results The stable overexpression of TRAIL was detected in Panc-1 cells transfected by Ad-TRAIL.Ad-TRAIL significantly inhibited cell viability of Panc-1 cells.Furthermore,co-culture of cancer cells transfected with TRAIL resulted in the nontransfected cell inhibition by bystander effect.Moreover,the percentage of apoptotic cells was significantly higher in the Ad-TRAIL-treatment group compared to the control groups(P

ObjectiveTo monitor the treatment effect of acute leukemia by multiparmeter flow cytomtry(MPFC), MethodsThe patientswith acute leukemia all achieved complete remission after remission induction therapy.Before the chemotherapy,the bone marrow cell morphology and MPFC inspection were used for each patient. Through statistical analysis, the relationship between morphological feature andrecurrence,as well as time duration till recurrence were obersved. ResultsThe 122 specimens from 30 patients were observed.The specimens were analyzed by morphological examination of bone marrow cells,and the other 61 specimen through MPFC. There were 8 positive specimen(13.11％)through morphological examination,while 18 positive specimen(29.51％ )through MPFC. There was a significant difference between the two groupes by x2 test (x2=4.33,P＜0.05).The 18 MPFC-positive specimens were from 10 patients.In the follow-up period, there were 6 patients whose morphological examination also prompted the recurrence in 1-4 months. Three patients were failed to follow up because of 2 patients became negative and 1 patient died. ConclusionMPFC is more sensitive and able to assess the effect of acute leukemia treatment more objectively and detect the recurrence earlier than morphological examination.

Objective To investigate the natural infection status of murine norovirus ( MNV) in laboratory mice in Shanghai area and isolate MNV from mouse cecal feces .Methods To collect cecal contents and serum samples from 319 specific pathogen-free ( SPF) mice coming from different research institutions .Reverse transcription-polymerase chain reaction ( RT-PCR) and enzyme linked immunosorbent assay ( ELISA) were used to detect MNV infection in the mice , re-spectively.The positive stool samples were diluted and filtered through 0.22 μm membrane, inoculated into RAW 264.7 cells, and then identified by RT-PCR.Results There were 95 positive results in the 319 cecal samples by RT-PCR, and the positive rate was 29.78%.Among 180 serum samples which were tested by RT-PCR, 70 samples were positive by ELISA, and the positive rate was 38.89%.The infected RAW 264.7 cells showed cytopathic effect ( CPE) within 72 h. After 3 times of freezing and thawing , RT-PCR obtained a 187 bp band.Conclusions The results from the present study show that there is a high natural infection rate of MNV in laboratory mice in Shanghai area , and the strict breeding manage-ment must be strengthened .

Objective To evaluate cerebrospinal fluid adenosine deaminase(CSF-ADA)activity in the diagnosis of tuberculous meningitis(TMB), and to observe its dynamic changes. Methods A total of 160 patients were included and were divided into two groups: 76 cases of TBM and 84 cases of non-TBM.Among the cases of non-TBM, there were 36 cases of bacterial meningitis, 30 cases of viral meningitis and 18 cases of cryptocoocal meningitis. All the patients were measured with their CSF-ADA activity by Enzymecoupled assay(Trinder method)and 47 patients of TBM were measured again after 2 weeks' and 6 weeks'antitubercular therapy. Results were expressed as(-x)± s. Mann-Whitney U test and paired-samples t test were used. Results CSF-ADA activity in TBM group was(12.9 ±6.4)U/L, while that in the non-TBM group was(6.0 ± 4.1)U/L, the difference was of statistical significance(U = 7.860, P ＜ 0.05). With the cutoff value of 9 U/L, the sensitivity and specificity to differentiate TBM from non-TBM was 84.21% and 83. 33%, respectively. CSF-ADA activity decreased in TBM patients after antitubercular treatment.Conclusions CSF-ADA activity can be an effective laboratory marker for early differential diagnosis of TMB with the cut-off value of 9 U/L. Dynamic changes of CSF-ADA activity may be a indicator for the effect of antitubercular treatment.

Objective To evaluate two kinds of anterior screw fixations in the treatment of odontoid fractures. Methods A total of 36 patients with D' Alonzo type Ⅱ odontoid process fractures were treated with anterior screw fixation in our department from 1999 to July 2009. There were 28 males and 8 females at mean age of 42.3 years (rang 17-59 years). According to time and surgery procedures, the patients were divided into Group A ( from 1999 to June 2005, n = 11 ) and Group B ( from June 2005 to July 2009, n = 25 ). Patients in Group A received anterior hollow screw fixation of the odontoid process monitored under G-arm or C-arm, while those in Group B received anterior screw fixation of the odontoid process assisted by Iso-C 3D navigation system. The operation time and blood loss in two groups were compared by Student' s t test and analyzed with SPSS 13.0 statistical software. X-ray examination was performed in all patients 3, 6 and 12 months after operation to observe fracture union and stability of the upper cervical spine. Results The operation time was ( 102 ± 12) min ( range, 77-148 min) in Group A and ( 104 ± 14) min ( range, 71-150 min) in Group B, with no statistical difference ( P =0.21 ). The blood loss was (465 ± 5) ml (range, 20-130 ml) in Group A and (42 ± 6) ml (range, 26-150 ml) inGroup B, with no statistical difference (P = 0.16). All patients received reexamination three months after operation, which showed no bony union or dislocation but average 40% restriction of neck rotation. One year after operation, 30 patients (83%) got fracture union and six ( 17% ) got fiber healing, with average 24% restriction of neck rotation. Conclusions There is no statistical significant differences between two groups in aspects of operation time, blood loss and fracture healing. But anterior screw fixation of the odontoid process assisted by Iso-C 3D navigation system can reduce exposure to radiation of both patients and surgeons. Furthermore, solid screws can be applied to augment the fixation intensity and thereby reduce the complications caused by non - union.

Objective To determine the effects of histone deacetylase inhibitor suberoylanilide hydroxamic acid(SAHA) on the cell proliferation and apoptosis of the human hepatic stellate cell line LX-2.The possible underlying mechanisms were also investigated.Methods The LX-2 cells were treated with SAHA in vitro.The morphology of LX-2 cells in different concentrations groups was observed by inverted microscope;the proliferation of LX-2 cells was measured by MTT assay;the Annexin V-FITC and PI staining was used to detect the apoptosis of LX-2 cells by flow cytometry and fluorescence microscope;the expression of α-SMA,collagen Ⅰ,acH3K9,acH3K14 and acH3K18 were detected by Western blot.Results The morphology change of LX-2 cells showed that SAHA inhibited the proliferation rate of LX-2 cells and in a dose dependent manner(P<0.05).The LX-2 cells were sensitive to SAHA along with time increasing,and in a time-dependent manner(P<0.05).Western blot showed that the expression levels of α-SMA and collagen-Ⅰ were significantly lower(P<0.05),on the contrary,the acetylation levels of acH3K9,acH3K14 and acH3K18 were significantly higher (P<0.05).Conclusions The increased acetylation of the histone acH3K9,acH3K14,acH3K18 and the lower expressed α-SMA and collagen-Ⅰ in LX-2 cells may be one of the mechanisms of SAHA.

Objective:To study the effects of endothelial progenitor cells(EPCs)on the osteogenesis of bone marrow mesenchymal stem cell (BMSCs)sheet-implant complex.Methods:EPCs were added to the BMSC sheets,and the expression of osteogenesis-relat-ed genes was examined by real time PCR.Cell sheets were wrapped around implants to construct cell sheet-implant complexes and the complexes were subcutaneously transplanted into SCID mice.The complexes were harvested 8 weeks after operation and observed by micro-CT and histological examination.Results:The BMSC sheet with EPCs showed higher expression of Runx2,ALP,BMP2 and VEGF in the in vitro test;higher bone volume ratio,greater amount of new bone tissue and higher expression of Runx2 and BMP2 in the in vivo test.Conclusion:EPCs can improve the osteogenesis of BMSC sheet-implant complex.

Methylmalonic aciduria (MMA) is a common inherited autosomal recessive disorder resulting from defects in the enzyme methylmalonyl CoA mutase (MCM, mut complementation group) or in the synthesis of the MCM cofactor adenosylcobalamin (cbl complementation groups). The defects in the mut complementation group accounts for the largest number of patients with isolated MMA. At least 200 mutations in the MUT gene on chromosome 6p12 have been identified in MMA patients until now. This study aimed to investigate the clinical characteristics of MMA and genomic variations in the MUT gene of Chinese patients. Genomic DNA was extracted from 18 patients who were diagnosed as having isolated MMA by gas chromatography/mass spectrometry (GC-MS), and from some of their parents as well. Amplification and direct sequencing of the MUT coding regions (exon 2-13) and their adjacent intronic consensus splice sites were performed in order to identify the disease causing mutations. In this group, six novel mutations in the MUT gene, c.424A>G (p.T142A), c.786T>G (p.S262R), c.808G>C (p.G270R), c.1323_1324insA, c.1445-1G>A and c.1676+77A>C were identified. p.T142A and p.G270R were respectively detected at a heterozygous level in one patient. Two previously reported mutations, c.682C>T (p.R228X) and c.323G>A (p.R108H) were also found in this study. In addition, six previously described single nucleotide polymorphism (SNP), c.636A>G (p.K212K), c.1495G>A (p.A499T), c.1595A>G (p.H532R), c.1992G>A (p.A664A), c.2011G>A (p.V671I) and c.1677-53A>G were identified. In this study, we updated the spectrum of MUT mutations and identified the main MMA-causing mutations in Chinese MMA patients.