Objective To explore the changes of hepatocyte proliferation in rats during the progression of nonalcoholic fatty liver disease (NAFLD). Methods Rat models of NAFLD were established by giving a fat-rich diet. The rats fed with normal diet were taken as normal control at the same stage during the study. These rats were sacrificed at week 4, 8, and 12 respectively and collected the liver tissues. The cell cycle and the changes of the S-phase cell fraction (SPF), proliferation index (PI) was measured by flow cytometry. The expression of proliferating cell nuclear antigen (PCNA) was detected by immunohistochemistry. Results There was no significant difference between NAFLD group and the control group at week 4, 8. But as compared with the control group, SPF and PI of 12-week group were significantly increased and PCNA labeling index were also higher. Conclusion Hepatic hyperplasia occurred after the nonalcoholic steatohepatitis was established in the rat models of NAFLD. Hepatic hyperplasia probably was the reaction of inflammation and necrosis, but whether it was the early incident of hepatocarcinoma should be paid attention.

To study the characteristic of the metacarpal fracture and the forensic investigation of severity of the injury.52 cases with metacarpal fracture were reviewed.The incidence and the location as well as the patterns of fracture were analyzed.The severity of the fracture were different with different mechanism.

Objective To study two new factor Ⅸ mutations Cys82Ser and Ile288Ser in vitro and research the molecular mechanism of haemophilia B. Methods PcDNA3. 1 ( - ) FⅨwt expression plasmid was prepared. The mutated FⅨcDNA expression plasmids, PcDNA3.1 ( - ) FⅨM1 (Cys82Ser) and PcDNA3. 1 ( - ) F Ⅸ M2 (Ile288Ser) were constructed by megaprimer method respectively. Transient expression experiments were performed using HEK293 cells transfected with the expression vectors containing the wild-type or the mutation recombinant cDNA. PcDNA3. 1 ( - ) was used as a blank control. The expression proteins were detected by ELISA, factor activity assay and flourescence stain. Results The results suggested that the two FⅨ gene mutations did not induce the reduction of the mutant FⅨ mRNA compared with the wild-type FⅨ mRNA. The FⅨ:Ag in culture media and cell lysate of wild type conduct were assigned as 100. 0%. The results of PcDNA3.1 ( - ) FⅨ M1 mutation protein were (27. 1 ± 5. 2)% and (99.4 ±4. 1)% respectively. For PcDNA3. 1( - )FⅨM2, the results were (5.3 ± 1.8)% and (31.7 ±2. 5)% respectively. The FⅨ: C in culture media of wild type conduct was also assigned as 100. 0%. Then the two types of mutant protein were ( 8. 5 ± 3.2 ) % and ＜ 1%, respectively. Immunofluorescence microscopy result suggested that the intensity of perinuclear spot was reduced in cells transfected with PcDNA3.1 ( - ) FⅨM2 while staining for PcDNA3. 1 ( - ) FⅨM1 was predominantely diffuse without perinnclear enhancement. Conclusions These results strongly suggest that the FⅨ Cys82Ser mutation protein is not been correctly folded, by any possibility. The mutation protein has secretion defect. The secretion dysfunction and the protein degradation intracellular are possiblely the molecular pathology of Ile288Ser mutant protein.

ObjectiveTo establish an effective method for F9 gene mutation detection by using high resolution melting ( HRM ) curve analysis.Methods Peripheral blood samples of 55 hemophilia B (HB) patients were collected from Shanghai Ruijin Hospital during January 2005 to June 2010.Genomic DNA was extracted from the peripheral blood.PCR amplification combined with sequencing was used to identify the F9 gene mutations in 40 patients.HRM assay was established on the 21 DNA samples with known mutations in exonl to exon7 of F9 gene.Mutation scanning of exonl to exon7 by HRM combined with direct sequencing of exon8 was used in the molecular diagnosis of 15 HB patients with unknown F9 gene mutations.ResultsF9 gene mutation was detected in each of the 40 HB patients by direct sequencing.By HRM,the different melting curve patterns were identified in 19 out of 21 cases.The detection rate was about 90％.Through mutation scanning of exonl to exon7 by HRM combined with direct sequencing of exon8,F9 gene mutations were detected in all the 15 HB patients with unknown F9 gene mutations.Thirty-four F9 gene mutations had been identified in the 55 HB patients.ConclusionsA new strategy of HB genetic diagnosis,scanning mutations of exonl to exon7 combined with DNA sequencing of exon8 of F9 gene,is established in this study.The new strategy is efficient and reliable.

Objective To investigate current status and problems of coagulation factor Ⅷ and Ⅸ assay in domestic laboratories so as to provide the reference for implementing the standardization and quality improvement.Methods A questionnaire survey was carried out in 76 laboratories,and quality control materials were distributed to 54 laboratories for activity assay.The questionnaire information was analyzed statistically.Test results of quality control materials were classified into three groups according to the reagents and the ranked grading analysis were used to evaluate the performance.Results This research was investigative study.The amount of sample was less than 30 per month in 72％ (52/72)of laboratories.The frequencies of calibration were different,and 33％ (24/72) of laboratories did not perform calibration in a different assay batch.39％ (28/72)of laboratories did not run internal quality control,and about 21％ (15/ 72) of laboratories just performed the normal level quality control.Individual laboratories showed a high cumulative CV (＞ 30％) of intemal quality control.For normal FⅧ and FⅨ control materials,the CV of results were 11.3％-18.2％ and 11.3％-17.9％ respectively as well as 15.3％-20.3％ and 19.5％-21％ for abnormal.Of the three groups,the proportions of laboratories which the FⅧ test results out with consensus were18％,24％ and 22％ as well as 20％,24％ and 28％ for FⅨ.Conclusions The key requirements for quality control of coagulation factors active assay remain to be addressed and implemented.The repeatability and comparability in some laboratories are not satisfactory to meet the clinical needs.With the purpose of promoting quality improvement,we need to develop guidelines,organize related training and establish a national external quality assessment scheme.

Objective To study the effects of bone marrow mesenchymal stem cells(BMSCs) transplantation combined with artesunate(Art) on rat model of liver fibrosis.Methods 20 rats from 120 SD rats were selected randomly as normal control group.10 rats from those were executed and extracted marrow to culture BMSCs.The other 90 rats were injected intraperitoneally with CCl4 to make liver fibrosis rat models.10 rats from the 90 rats were selected randomly to ensure the liver fibrosis model was made successfully after 10 weeks.Then,the rest 80 rats were divided randomly into 4 groups:model group,Art treated group,BMSCs transplantation group and Art + BMSCs transplantation group.Fibrous degeneration of the hepatic tissue was detected by Masson stain.The mRNA expression of α-SMA,TGF-β1 in liver were detected by RT-PCR.The hydroxyproline(Hyp) level in liver was detected by enzyme digestion method.Serum TNF-αand TGF-β1 were detected by ELISA.Serum hepatic fibrosis parameters were detected by chemiluminescence and serum liver functional parameters were detected by biochemistry method.Results Liver functional parameters of Art + BMSCs transplantation group were better than those of the other groups.ALB in Art + BMSCs transplantation group was (29.13 ± 3.48) g/L,which was significantly higher than (26.31 ± 2.42) g/L in Art treated group (24.35 ±3.12)g/L in BMSCs transplantation group and (20.14 ±2.86) g/L in model group.There were significant differences among the 4 groups(q =6.27 ～ 19.01,all P ＜ 0.05).Liver fibrosis parameters of Art + BMSCs transplantation group were better than other groups.Hyp in Art + BMSCs transplantation group was (1.03 ±0.16) μg/L,which was significantly lower than (1.56 ± 0.19) μg/L in Art treated group,(1.45 ± 0.24) μg/L in the BMSCs transplantation group and (3.57 ±0.91) μg/L in the model group.There were significant differences among the 4 groups (q =20.47 ～ 27.70,all P ＜ 0.05).The expression of TGF-β1 mRNA of Art + BMSCs transplantation group was lower than other groups.The index of TGF-β1 mRNA expression in Art + BMSCs transplantation group was (20.16 ± 5.21),which was lower than (58.15 ±9.16) in Art treated group,(46.31 ±6.82) in the BMSCs transplantation group and (97.08 ± 14.23) in the model group.There were significant differences among the 4 groups (q =20.35 ～ 44.93,all P ＜ 0.05).Conclusion There're synergistic effects between Art and BMSCs in the treatment of liver fibrosis.

Objective To evaluate the effect and tolerance of late-course three dimensional conformal radiotherapy(LC3DCRT) combined with concurrent chemotherapy for stage Ⅲ non-small cell lung cancer(NSCLC).Methods From May 2000 to May 2003,48 such patients were entered into this study.The patient's characteristics were: 38 male and 10 female,with median age of 62 years(range 40 to 74);Karnovsky performance score ≥70;stage ⅢA 16 and ⅢB 32,squamous cell carcinoma 38 and adenocarcinoma 10.The treatment regimen consisted of conventional radiotherapy first(40Gy/20f/4W),followed by 3DCRT(24-30Gy/4-5f/2W) combined with concurrent chemotherapy.Conventional irradiation field encompassed the primary lesion,ipsilateral hilum and mediastinal lymph drainage region.LC3DCRT focused on the primary lesion only,with the 80%-90% isodose curve covering the planning target volume(PTV) and the target dose was prescribed to PTV.Supraclavicular metastatic lymph node was treated by mixed 6MV X-ray and electron beam to a total dose of 65-70Gy.Chemotherapy treatment regimen consisted of isophosfomide(25mg/m~2,d1、8,iv) and cisplatin(30mg/d,d1-3,iv) in the 1st and 5th week.Results Before the end of the second month after treatment,the complete response(CR)and partial response(PR) rate was 16.7% and 75.0%,respectively,with a CR+PR rate of 91.7%.The 1-,2-and 3-year local control and overall survival rates as monitored by the Kaplan-Meier method was 87.5%,50.0%,35.7% and 87.5%,46.7%,28.6%,respectively.All patients completed the planned treatment without interruption.Hematological toxicity and radiation-induced pneumonitis as shown by the WHO staging system were the most common acute toxicities but they were tolerable,with 8.3% of grade 3 leukopenia and 4.2% of grade 3 radiation-induced pneumonitis.The severity of the other acute toxicities such as nausea,fever,hemoglobin decrease,and radiation-induced esophagitis were mainly grade 1 or grade 2.Conclusions Late course three dimensional radiotherapy combined with concurrent chemotherapy shows a promising results with tolerable acute toxicities.Long-term survival and late toxicities need further observation.

BACKGROUND:Human periodontal ligament stem cels are a kind of mesenchymal stem cels that have self-renewal and multidifferentiation potential. Previous studies have showed that human periodontal ligament stem cels can differentiate into osteoblast-like cels or adipocyte-like cels under appropriate induction. Yet few studies have focused on the expression level of parathyroid hormone 1 receptor which wil affect the osteogenic potential of Human periodontal ligament stem cels. OBJECTIVE:To examine the expression level of parathyroid hormone 1 receptor between human periodontal ligament stem cels and human periodontal ligament cels and to discuss the role of parathyroid hormone 1 receptor in osteogenic differentiation. METHODS:By using magnetic-bead cel sorting, we separated and identified the human periodontal ligament stem cels and human periodontal ligament cels. We examined and compared the mRNA expression level of parathyroid hormone 1 receptor in human periodontal ligament stem cels and human periodontal ligament cels by Real-Time PCR. Osteoblastic differentiation was examined throughin vitro matrix mineralization by alizarin red staining and alkaline phosphatase assay. RESULTS AND CONCLUSION: Positive immunomagetic sorted cels were positive for STRO-1, CD146, Vimentin, indicating that they were periodontal ligament stem cels. Parathyroid hormone 1 receptor was expressed in human periodontal ligament stem cels and mainly located in cel membrane and cytoplasm which were similar to human periodontal ligament cels and MG63 cels. The expression of parathyroid hormone 1 receptor in human periodontal ligament stem cels was 3.7 times higher than that in human periodontal ligament cels, which was similar to that in MG63 cels. After osteogenic induction, human periodontal ligament stem cels showed a higher expression of parathyroid hormone 1 receptor and osteoblast-related genes as wel as the activity of osteoblast alkaline phosphatase and mineralization compared to human periodontal ligament cels. Our data showed that parathyroid hormone 1 receptor was higher in human periodontal ligament stem cels than human periodontal ligament cels and the expression was related with osteogenic differentiation, suggesting that human periodontal ligament stem cels display a higher potency of osteogenic differentiation and act as seed cels with a vast application prospect in oral tissue engineering.

Objective To investigate the genetic diagnosis and molecular pathogenesis of four patients with combined deficiency of coagulation factor Ⅴ and Ⅷ and their family members. Methods The APPT, FT, FⅤ: C, FⅧ: C were detected for phenotypic diagnosis. Thrombin generation assay was applied to determine the generation condition of thrombin in patients and healthy controls. Cenomic DNA was extracted from peripheral blood using the TianGen RelaxCene Blood DNA System;amniotic fluid DNA was extracted with phenol-ethyl ether method. The LMAN1 and MCFD2 genes were analyzed by PCR. Gene mutations were detected with nucleotid sequences by using end-labeling dideoxy method. Results The APTT of Proband 1 was significantly prolonged to 88. 2s and her PT was prolonged to 19. 6 s. The combined deficiency was identified with FⅧ (FⅧ: C 24. 2% ) and FV(FⅤ: C 9. 1% ). Proband 2 and 3 were sisters. The coagulation studies revealed that both of them had prolonged APTT (71.6 s and 74.6 s respectively) and PT (22. 1 s and 18. 3 s respectively). The combined deficiency of FⅤ (FⅤ: C 7. 6% and 14. 5% respectively) and FⅧ( FⅧ: C 25% and 19.6% respectively) were identified. Proband 4 was detected to have the prolonged APTT (70.3 s),PT (18.2 s) and the deficiency of FⅤ(FⅤ: C 9. 4% ) and FⅧ (15. 7% ). The remaining phenotype indicators test of the 4 probands were normal. The diagnosis for the 4 probands was combined deficiency of factor Ⅴ and Ⅷ. The proband 1 was detected to have compound heterozygous mutations in LMAN1 gene while having the LMAN1 and MCFD2 direct gene sequencing. One mutation was a small insertion located on exon 8 [ nt912insA (X71661. 1)] that resulted in p. 305frameshiftX20 and her mother was detected to have the same heterozygous mutation on the the locus. The other mutation was located on exon 11: nt1366C > CT ( X71661. 1 ) , p. 456Arg > Stop which was inherited from her father. Amniocyte DNA was detected to have only one heterozygous mutaion [nt1366C > CT (X71661. 1) , 456Arg > Stop] inherited from the father. No mutation in MCFD2 gene was found in proband 1 and her parents. The analysis of the MCFD2 gene in proband 2 and 3 revealed a novel homozygous single base substitution (nt411T>C) in exon 4, which results in the exchange of the amino acid isoleucine by the amino acid threonine at amino acid position 136 (p. Ile136Thr). Sequencing of the whole LMAN1 gene showed that the proband 4 had one homozygous nonsence mutation in the exon 5 of the LMAN1 ( nt615C >T,p. 202 Arg> Stop). All of the 4 probands with combined deficiency of FⅤ and FⅧ showed declined endogenous thrombin potential in the thrombin generation tests. Conclusion The combined deficiency of FⅤ and FⅧ in the proband 1 results from the compound heterozygous mutations ( nt1366C > CT and nt912insA) in LMAN1 gene, which are inherited from her parents respectively. The prenatal genetic investigation for the patient mother with preganency indicates that the fetus is a female carrier with one mutation (nt1366C > CT) inherited from the father. The homozygous missence mutation ( nt411T > C, p. Ile136Thr) in the MCFD2 gene accounts for the proband 2 and 3. The daughter of the proband 2 is a carrier with a heterozygous mutation inherited from her mother. The homozygous nonsence mutation in the LMAN1 gene of the proband 4 results in the deficency of F Ⅴ and FⅧ.

objective To make genetic diagnosis in two haemophilia families with recombination. Methods For hemophilia A(HA)family,screening of the F Ⅷ intron 22 and intron 1 inversion mutations was employed to identify the mutation. Linkage analysis with 8 polymorphic markers was adopted in the pedigree. For hemophilia B(HB)family,DNA sequencing of all coding regions of FⅨ gene Was used to detect the mutation directly. The muhifluorescent PCR method employing six FⅨ related STR was adopted in linkage analysis.Results In the HA family,the proband was positive in inversion 1 detection and the relative female was inversion 1 carrier. But linkage analysis with polymorphic markers showed contrary resuhs. Some markers certified that the female inherited the disease chromosome of the family while the others showed contrary results.In the HB family,it was unsuccessful in sequencing the exon 7 of the F Ⅸ gene in the proband and there was no mutation found in the other parts. The relative female and her amniocyte DNA were successful in sequencing the whole F Ⅸ gene and no mutation was detected.The linkage analysis of the family showed contrary results. Recombination occured in these two families. Conclusions Although the linkage analysis iS convenient and effective in carrier and prenatal diagnosis of hemophilia families. The recombination risk shouldn't be neglected especially when the polymorphic markers give inconsistent information for linkage analysis. It is necessary to find some high inforrnative markers intragenic or on the telomeric side to the gene in order to prevent the risk of recombination.