Objective To verify the targeting regulatory relationship between microRNA (miR)-21-5p and mitogen-activated protein kinase kinase 3 (MKK3).Methods miR-21-5p and MKK3 targeted relationship was analyzed by bioinformatics database predict.Constructing MKK3 3'UTR reporter vector (MKK3-3U) and mutation reporter vector (MKK3-3 U-M) was constructed by synthesizing miR-21-5p mimics (mimic),miR-2 1-5p inhibitor (inhibitor),nonsense miR (NC).NC (NC1 group),mimics (mimic1 group),inhibitor (inhibitor1 group) together with MKK3-3U cotransfected human embryonic kidney (HEK293) cells,respectively.Empty vector-pYr-MirTarget (NC2 group),MKK33U (Mt group),M KK3-3U-M (Mut group) together with mimic co-transfected HEK293,respectively.The fluorescence ratio was detected by dual luciferase assay.NC,mimics,inhibitor intervened human renal tubular epithelial (HK-2) cells,respectively.RT-PCR and Western blot were used to test cell MKK3 expression level in HK-2.Results 1.The fluorescence ratios of NC1 group,mimicsl group,and inhibitor1 group were 100.00％,67.99％,133.17％,and there was difference between mimic1 group and NC1 group(t =19.93,P ＜ 0.05).2.The fluorescence ratios of NC2 group,Mt group and Mut group were 100.00％,65.30％,98.48％,and there was difference between Mt group and NC2 group (t =14.39,P ＜ 0.05).There was no significant difference between NC2 and Mut group (t =0.63,P ＞ 0.05).3.MKK3 mRNA relative expression levels of the control group mimic2 group and inhibitor2 group were 1.36 ± 0.02,1.01 ± 0.04,1.43 ± 0.06 ; relative protein expression levels were 0.97 ± 0.05,0.62 ± 0.06,1.36 ± 0.32.MKK3 mRNA and protein levels of mimic2 group were significantly lower compared with the control group (F =85.98,118.26,all P ＜ 0.01).Conclusions MKK3 is the target gene of miR-21-5p.miR-21-5p can regulate the expression of MKK3 both in mRNA and protein levels.miR-21-5p may be an important regulatory molecule in p38 mitogen-activated protein kinase signaling pathway.

Objective To establish a noninvasive method for prenatal genetic analysis by using maternal serum and apply the method in fetal sex determination,paternity testing. Methods Samples of maternal serum from 53 pregnant women (11 to 36 weeks of gestation) were collected. The DNA extracted from each sample was amplified by using"Y-PLEX 6" amplification kit .which enabled the simultaneous analysis of six Y-STR loci including DYS393.DYS19.DYS389 II, DYS390, DYS391 and DYS385. The PCR products were detected by using ABI PrismTM 377 Sequencer and genotyped by related analysis software. Results (1) Y-STR specific alleles were detected in the maternal sera of all 29 mothers bearing male babies. Among the six Y-STR loci,specific alleles were detected in 29/29 at DYS393 locus,in 18/29 at DYS19 locus and in 10/29 at DYS390 locus. (2) Y-STR specific alleles were not detected in maternal sera of 24 pregnant women bearing female babies. (3) According to the presence of specific alleles at DYS393 locus and the value of allelic peak height and peak area, the accuracy of fetal sex determination was 100% . (4)The observed Y-STR alleles of each prenatal specimen from pregnant women with male fetuses were the same as the results of their husbands. Conclusion The assay of highly polymorphic Y-STR genotyping system developed by the authors provided a sensitive, accurate and non-invasive method to prenatal diagnosis. Our results demonstrate that fetal sex can be accurately determined and imply that paternity testing could be performed for pregnant women carrying male fetuses.

Objective To study the molecular polymorphism and the distribution of HLA-B27 subtypes in southern Chinese Han patients with Ankylosing Spondylitis and healthy controls.Methods A total of 46 samples form southern Chinese Han patients with AS and 80 non-related blood samples from healthy peripheral blood stem cell donors with B27-positive identified by rSSO Lumminex flow array assay were subjected to sequencing analysis of exon 2 ,3 and 4 of HLA-B gene by the sequence-based typing,the purified products of sequencing reaction were electrophoresed on ABI 3730 DNA sequencer and the designation of HLA-B27 allele was accomplished using the Assign3.5 software. The ambiguities and the detected "rare" alleles were confirmed using the PCR-SSP commercial kit. Results In the 46 B27-positive patients with the diagnosis of AS,four alleles,namely B2704,B2705,B2707 and B2724 were determined. The frequencies for these four alleles were 82.98%(39/47),12.77%(6/47),2.13%(1/47) and 2.13%(1/47),respectively. In the 80 B27-positive control individuals,seven B27 related alleles were identified. The frequency for the two dominant subtype B2704 and B2705 were 57.32%(47/82) and 26.83%(22/82),respectively. Both the B2706 and B2707 were observed 5 times with a frequency of 6.10%(5/82),three alleles B2703,B2715 and B2724 were detected only once with a frequency of 1.22%(1/82).Conclusion Our study shows that HLA-B2704 and B2705 were the predominant subtypes in normal healthy controls,however,B2704 was the predominant subtype for the AS group in southern Chinese Han patients.

Objective Mutations of 13 CODIS (Combined DNA Index System) STR core loci in 532 cases of paternity testing were observed in confirming paternity, the mutation rate and the mutation type were studied. Methods 587 cases of paternity testing were routinely carried out using AmpFe STR Profiler Plus and Cofiler PCR Amplification Kits. When one or two STR exclusions were found, then HLA system and other blood groups were tested by molecular typing, and sixteen STR loci were genotyped by using PowerPlexl6 PCR Amplification Kit. If necessary, the genotyping of Y chromosome specific STR and HLA allelic sequencing were added. Results 1052 meiosis were observed among the 532 cases in confirmed paternity, 18 mutation events were found in 17 paternity cases. Single-locus mutation was observed in 16 cases, and mutation at two STR loci was observed in one case. The observed mutational loci include: D5S818, D3S1358, D16S539, CSFIPO, D21S11, D13S317, D7S820, vWA, D18S51 and FGA. The mutation rates for D18S51 and FGA loci were both 0.29% , which were the highest among the ten mutational loci. 11 events of paternal source mutations, 5 events of maternal source mutations and two events of indistinguishable mutations were observed in 18 STR mutational events. Conclusion When one or two STR exclusions were found in paternity testing, other more genetic markers must be detected as complement before making final conclusions.

Objective To compare the curative effects of intertrochantefie fractures in aged by two operative methods. Methods 85 cases of elderly patients suffered from intertrechanteric fractures were retrospectively ana-lyzed. Out of the 85 cases,45 cases were treated with dynamic hip screw(DHS) and 40 cases were fixed with Gamma nails. The curative effects and complications were compared simultaneously. Results The total excellent rate was 83.5% in 85 patients,the rates of DHS and Gamma nails group were 77. 8% and 90% (X2 = 2. 84, P 0. 05), re-spectively. The operative time (66. 4 ± 19. 4)min in DHS group were less than the Gamma nail group (875 ± 25.5) rain (t=2. 451 ,P < 0. 05), but the length of surgical incision (19. 6± 5. 1)cm greater than the Gamma nail group (10. 3 ± 4. 7) cm(t = 2. 501, P < 0. 05). The complications of DHS group (11.1%) were significanfly higher than that of Gamma nail group(2. 5%) (X2 =3.94,P<0. 05). Conclusion As long as the physical condition can toler-ate,surgery recommend the use of DHS and Gamma nail surgical techniques in aged patients with intertrechanteric fractures.

Objective To develop an efficient method for detecting the short tandem repeat(STR) of fetal DNA in maternal plasma by miniSTR technique.Methods A total of 9 blood samples form pregnant women from 11 to 27 weeks of gestation were collected.Each isolated total plasma DNA was amplified in single multiplex using the ABI MiniFilerTM kit,which could simultaneously genotype the 9 miniSTR loci,including D13S317,D7S820,D2S1338,D21S11,D16S539,D18S51,CSFIPO,FGA and Amelogenin,and the PCR products were detected by using ABI PrismTM 3100 DNA Sequencer.The allelic designation of each STR locus was accomplished using the GeneMapper ID 3.2 software.Results Father-origin fetal STR allele was detected in all the 9 plasma DNA samples.An average of 3.1 fetal STR alleles of the 8 autosomal STR loci was observed in each of the 9 plasma DNA samples.As for the Amelogenin locus,Amelogenin Y allele was detected in 5 plasma DNA samples from pregnancies with male fetus,and allelic peak height values were all over 50 RFU,according to ABI Mini FilerTM PCR conditions,and the ratio of Amelogenin Y allele peak height value to Amelogenin X allele peak height value was 8.45%.However,no Amelogenin Y allele was detected in other 4 plasma DNA samples from pregnant women with female fetus.Conclusion The miniSTR technique is suitable for STR genotyping using fetal DNA in maternal plasma,and it suggests a broad application in noninvasive molecular prenatal diagnosis.

Objective To research the influence of autoclave sterilization on elastic module and flexural strength of five types of posts.Methods Shidelong carbon,glass and quartz fiber post,tenax glass fiber post and Anthogyr titanium post were chosen,and all the samples were divided into two groups which included five subgroups according to the model of posts,each subgroup including six samples.Experimental groups were autoclave sterilized(50 min at 134℃ and 2.2MPa),While the control groups were not.With three point loading system,elastic module and flexural strength of all the samples were measured.Results were statistically analyzed to find the difference between them.Results There was no statistical difference between Shidelong carbon,glass,quartz fiber post and Tenax glass fiber post in elastic module and flexural strength in both experimental group and control group,while Anthogyr titanium post gained the highest elastic module and flexural strength.Paired-samples t test also showed there was no statistical difference between experimental group and control group in elastic module and flexural strength.Conclusion Fiber posts perfectly meet the need of clinical use on flexural strength.Furthermore,with elastic module similar to dentin and much lower than metal posts,fiber posts will help to protect the remaining tooth structure from fracture.Autoclave sterilization has a little influence on elastic module and flexural strength of posts,which allows practitioners to systematically sterilize the posts in clinic.

BACKGROUND:During the research of ABO blood type antigen, the overwhelming majority samples of same ABO gene express a normal and same ABH antigen. But a certain amount samples with the same ABO genetic background show different antigen intensity expression as for different family or individuals. The ABO blood type has complex expression regulation mechanism. Analysis of ABO blood group serology and genetic background of these rare bi-specific AB phenotype specimens, and further studying on epigenetics may partly revealed ABO gene expression mechanism. OBJECTIVE:To study methylation of CpG island and explore the relationship between ABO gene promoter coding glycosyltransferase with dual donor specificity and ABH antigen expression. METHODS:Six samples detected as CisAB or B(A) phenotype were studied in this paper. The whole code sequences and promoter sequence of ABO gene were amplified respectively. The level of CpG methylation in promoter of ABO gene was further detected with bisulfite treatment method. RESULTS AND CONCLUSION:Among the six bi-specific AB phenotype samples, two previously-identified CisAB05/B(A)06 al eles with nt803C>G on the basis of B101 al ele sequence could be seen, and three additional methylated sites nt-33(30%), nt+27(50%) and nt+49(50%) were found between the two regions of CpG island in promoter of ABO gene. Two CisAB01 al eles with nt803C>G mutation on the basis of A101 sequence were found at nt-26C(10%). Other two B(A)04 al eles contained nt640A>G mutation on the basis of B101 sequence were found in the whole code sequences regions, and six additional methylated sites nt-33(10%), nt+16(50%), nt+57(60%), nt+59(60%), nt+68(60%) and nt+74(60%) were found between the two samples. No abnormity was identified in the promoter region of ABO gene. Our results indicated that the differential methylation levels in the CpG island of ABO gene promoter region may affect ABH antigens expression on the red cel membrane even if the samples had the same ABO genetic background.

BACKGROUND: Umbilical cord blood (UCB) with limited karyocytes is mainly used in child patients. Recently, physicians have tried to mix two units of cord blood in the treatment of adults with hematological system diseases.OBJECTIVE: To monitor quantitatively the dynamic changes and the development rules of engraftment, chimera types and relative amount after allogeneic transplantation of mixed UCB from two units in adults with leukemia.DESIGN: Donors and the recipient were regarded as observational subjects in umbilical cord blood transplantation (UCBT). DNA extracted from blood samples of donors and the recipient before and after transplantation was considered as detecting samples. Short tandem repeat (STR) loci were as observational measures.SETTING: Key Laboratory of Immunology and Genetics of Institute of Transfusion Medicine of Shenzhen Blood Center.PARTICIPANT: A 43-year male patient with acute myeloid leukemia (AML), 75 kg, who was hospitalized at Shenzhen Hospital of Peking University, was enrolled in June 2005. The patient received two units of human leucocyte antigen (HLA), one locus mismatched unrelated UCBT (2.5×107 kg-1 karyocytes in UCB 1, and 1.53×107 kg-1 karyocytes in UCB 2) at month 6 after complete remission from first chemotherapy. UCB was collected from Guangzhou umbilical cord blood bank. The patient signed the informed consent.METHODS: The adult with AML received two units of HLA, one locus mismatched unrelated UCBT (2.5×107 kg-1 karyocytes in UCB 1, and 1.53×107 kg-1 karyocytes in UCB 2). Nine STR loci of the blood sample were determined before and after transplantation by quantitative technique of fluorescence labeling with multiplex polymerase chain reaction (MPCR), while the engraftment and chimera types were qualitatively evaluated by comparing differential loci between the recipient and the donors. The relative amount of two units of UCB was calculated in the patient after transplantation according to the differential gene peak areas of two donors with 377XL DNA sequencer after fluorescence scanning. The engraftment level and the development rules of donors' cells were analyzed quantitatively. In addition, the results were also compared with that of HLA loci distinct analysis for engraftment.MAIN OUTCOME MEASURES: After UCBT, transition process of nine STR loci of the recipient and two donors was observed, and engraftment was quantitatively and qualitatively described.RESULTS: Two units of UCB at day 15 after transplantation were engrafted simultaneously and revealed a complete chimera of the two. The relative amounts of UCB 1 and UCB 2 were 51.3% and 48.7%, respectively. Subsequently, UCB 1 went up to 70.0% and UCB 2 declined to 30.0% at day 30. However, only the genotype of UCB 1 was detected at day 52, and engraftment turned to a complete chimera of a single donor. The one with fewer karyocytes was rejected and the one with more karyocytes was engrafted for a long term.CONCLUSION: To detect quantitatively STR chimera with fluorescence labeling and MPCR can show precisely the engraftment level and the change of two units of UCB. It provides an accurate and reliable experimental basis for clinical UCB application and donor selection. It is proved that adult transplantation at the same time with mixed UCB from two units HLA one locus mismatched unrelated donors is feasible.

Objective To study curative effect of core decompression and autologous bone marrow stem cell transplantation combined with traditional Chinese medicine in the treatment of early femoral head necrosis (ANFH).Methods 30 patients with femoral head necrosis treated by different methods were divided into 3 groups: core decompression A group) 10 cases(12 hips) ;core decompression pus stem cells transplantation(B group) 10 cases(13 hips) ;core decompression pus stem cells transplantation combined with traditional Chinese medicine(C group) 10 ca-ses( 10 hips) ;The X-ray 、CT MRI、Harris score( HHS) .curative effect were observed.Results The phase of Ⅰ,Ⅱ,Ⅲ,in 3 groups didn't appear deformation and collapse at 3,6,12 months;The score of Harris after 12 months 93 points were higher than that preoperatively 57.5 points(χ 2= 5.81 ,P<0.05) ;The signal ratio of femoral head volume in MRI was 42% before treatment,disappearance of femoral head necrosis after treatment;Total curative effect of B,C group,werehighter than that Agroup(χ2 =3.81,χ2 =3.98,P<0.05).Conclusion The operative treatment of ANFH with core decompression and stem cells transplantation combined with traditional Chinese medicine had the advantage of minimal damage,simplicity,accuracy,and effectiveness.