Objective To explore clinical effect of reconstruction for chronic Achilles tendon rupture of Kuwada IV type with flexor hallucis longus (FHL) harvested using a minimally invasive technique.Methods The data of 35 patients with chronic Achilles tendon rupture of Kuwada IV type was retrospectively analyzed who were treated by FHL which was harvested using a minimally invasive technique from July 2006 to June 2011.There were 21 males and 14 females,with the age from 23 to 71 years (average,42.1 years).All patients were unilateral injury.MRI showed Achilles tendon rupture fissures 6.0-9.2 cm.The local appearance and function recovery on postoperation was observed,and all patients were assessed with the American Orthopaedic Foot and Ankle Society (AOFAS) ankle-hindfoot score and Leppilahti Achilles tendon repair score.Results Thirty-two patients were followed up for 18 to 72 months,with an average of 33.2 months.Except for 1 patient whose wound healed after six weeks through resuture immediately for the wound dehiscence occurred in the ten days,other patients' wound healed smoothly.The average of AOFAS ankle-hindfoot score had increased from 51.92±7.08 preoperatively to 92.56±6.71 postoperatively.Leppilahti Achilles tendon repair score had increased from 72.56±7.43 preoperatively to 92.58±5.1 postoperatively.Twenty-seven cases were excellent,good in 3,and fair in 2,with the total excellent and good rate 93.8％ (30/32).No case of the sural nerve and tibial nerve injury,plantar painful scar,plantar outside nerve injury.MRI of Achilles tendon showed even signal without signal of tear and cystic degeneration.Conclusion Reconstruction for chronic Achilles tendon rupture of Kuwada IV type with FHL harvested using a minimally invasive technique offers a desirable outcome in rapid postoperative recovery,high strength in tenodesis,fewer complications.

Objective To analyze the clonal gene rearrangement and complementarity determining region 3 (CDR3) repertoire of TCR β-chain in fine needle aspiration biopsy (FNAB) specimens of lymph node metastasis in patients with breast cancer. Methods The TCR CDR3 region genes of 24 TCR Vβ subfamilies were amplified by utilizing RT-PCR technology, and the CDR3 lengths of TCR β-chain were analyzed with gene scan technology for 2 cases with lymph node reactive hyperplasia and 3 patients with lymph node metastasis of breast cancer. The clonality of T cells presumed by spectra typing was further confirmed by CDR3 sequencing. Results TCR β-chain presented specific repertoire skewing in metastatic lymph node,and only 3-5 TCR Vβ subfamily of T cells were identified, respectively. Clonal expanded T cells, including oligoclonal, polyclonal patterns, in one or more Vβ subfamilies were found in all cases. The oligoclonal expanded T cells had different CDR3 amino acid sequences. Conclusion There are characteristic T cells cloning proliferation and selected usage of TCR Vβ subfamily T cells could be found in metastatic lymph node.The sequences of CDR3 in different TCR clone proliferation are mostly different.

The pivotal characteristics, the current situation, the challenge being faced with and the potential solution related to the automatized flow line of the clinical laboratory is introduced in order to provide the reference for the healthy development of the full automatization of the clinical laboratories in China.

Objective To construct the eukaryotic expression vector for mouse microRNA miR-21 and identification its expression activ-ity in 293 cells. Methods The genomic sequence containing pre-miR-21 was amplified from mouse genomic DNA by PCR and cloned into the pRC/CMV plasmid. The constructed recombinant plasmid pRC/CMV-mmu-miR-21 was transfected to 293 cells by lipofectamine 2000, and the stably transfected cells were screened with G418,from which total RNA was extracted for detecting the expression of mature miR-21 by northern blot. In the meantime,a luciferase report plasmid examing the activity of miR-21 named pmiR-21-Luc reporter was also construc-ted,and luciferase activity analysis indicated the product of pRC/CMV-mmu-miR-21 indeed had biological activity. Results Both restriction enzyme digestion analysis and sequencing proved the recombinant plasmids were constructed correctly. The miR-21 was highly expressed in the screened clones of 293 cells and it had good biological activity. Conclusion The eukaryotic expression plasmid of mouse miR-21 was successfully constructed,which laid the foundation of further investigation of the role of miR-21 during skin wound healing.

AIM: To optimize the extraction process for coumarins in Angelica Dahurica by supercritical CO 2 as guided by the contents of oxyimperatorin, imperatorin and the total coumarins in the extract. METHODS: HPLC was applied to determine the contents of oxyimperatorin and imperatorin. orthogonal design was applied to optimize SFE process. Range and variance analysis as well as stepwise nonlinear regression analysis were applied to deal with experimental results. RESULTS: The optimum process was established as following: 21MPa as extracting pressure, 50℃ as extracting temperature, 3.5h as extracting time, 20 mesh as pulverized degree and 6.5MPa as separating pressure. The relative error between experimental data and calculated value was less than 5%. CONCLUSION: Changes in extracting pressure, temperature, time, pulverized degree would affect the extracting results remarkably. Some factors had interactions on the extraction of coumarins in Angelica Dahurica . Regression equation was reasonable and could forecast results precisely.

Objective To observe the expressions of small heterodimer partner (SHP) in rat model of hepatic fibrosis and its role in fibrosis.Methods A total of 30 healthy male SD rats were divided into two groups: 6 rats in control group and 24 rats in model group.The model group was further divided into four subgroups which were sacrificed at different time points,2,4,6 and 8 weeks after intraperitoneal injection of dimethylnitrosamine (DMN).After establishment of the rat model,blood samples and liver tissue specimens were collected at week 2,4,6 and 8 respectively.The sections of liver tissue were stained with HE and Masson and then were observed under optical microscope.The expressions of SHP mRNA and protein were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot,respectively.The comparison of means among the groups was performed by univariate ANOVA.Results The hepatic fibrosis was most obvious at 4 weeks and 6 weeks after the intraperitoneal injection.In model groups,alanine aminotransferase (ALT) and aspartateaminotransferase (AST) levels gradually increased and reached a peak at week 4,which were (169.2±16.2) and (193.3±31.1) U/L,respectively.Meanwhile,albumin level in the model group decreased gradually,reaching the nadir at week 6,and the differences were statistically significant at all selected time points between the model group and control group (FAST =83.10,FALT =104.63,FAlb =54.24; all P＜0.05).After modeling,the expression of SHP mRNA and protein in model group were significantly increased than those in control group,and reached a peak at week 4 (0.4494±0.0555 and 1.1155 ±0.1546,respectively),then both mRNA and protein levels decreased gradually at week 6 and 8 which obviously lower than the control group.Transforming growth factor (TGF)β1 mRNA expression level also increased gradually,reaching a peak at week 4 (0.9625±0.1196),and the differences between model subgroups and control group were statistically significant (F=25.740,171.383,118.393 and 94.343; all P＜0.05).Linear correlation analysis showed that SHP mRNA was positively correlated with TGFβ1 mRNA (r=0.593,P＜0.01).Conclusion During the progression of hepatic fibrosis,the SHP expression increases at the beginning and then turns to decrease,which suggests that SHP may play an important role in the development of hepatic fibrosis.

Objective To observe the dynamic expressions of kielin/chordin-like protein (kcp) in mouse model of hepatic fibrosis and the effects of bone morphogenetic protein-7 (BMP-7)intervention on the expressions,and to explore a new target for treatment of fibrosis.Methods A total of 50 healthy male ICR mice were divided into three groups:control group(n=10) ; model group (n=30) and BMP-7 treatment group (n=10).The model group was further divided into three subgroups according to different time points:subgroups of 4,8 and 12 weeks with 10 mice in each subgroup.The mouse model of hepatic fibrosis was established by hypodermic injection of carbon tetrachloride(CCl4 ).The mice in BMP-7 treatment group began to receive human recombmant BMP- 7via intraperitoneal injection after 8 weeks of the first administration of CC14 and lasted for 4 weeks.The serum levels of alanine aminotransferase (ALT),aspartate transaminase (AST) and albumin (Alb) were detected.The pathological changes of liver were observed under optical microscope after HE and Masson staining.The dynamic expressions of kcp mRNA and protein of each group were detected by reverse transcription-polymerase chain reaction,immunohistochemistry and Western blot.The comparison of means among groups was done by univariate ANOVA.Results In model group,ALT and AST levels increased,while Alb level gradually decreased,and peaked at week 12.BMP-7treatment could reduce the changes,and there were significant differences among groups (F=23.501,34.600 and 16.244,respectively; all P＜0.05).In normal control group,the expressions of kcp mRNA and protein were low,while those in model group were gradually increased.BMP-7 treatment could achieve remission and the changes were all significantly different among groups (F=30.362 and 10.727,respectively; P＜0.01 or 0.05).The expression of kcp mRNA was positively correlated with levels of transforming growth factor (TGF)-β1 mRNA and BMP-7 mRNA (r=0.760 and 0.769,respectively; both P＜0.05).Conclusions BMP 7 can improve the hepatic fibrosis in mice.kcp may play an important role in the occurrence and development of liver fibrosis which is a potential therapeutic target for hepatic fibrosis.

ObjectiveTo investigate whether CD4+ CD25+ CD127dim/- regulatory T lymphocytes (Treg) can induce the proliferation of hepatic stellate cells (HSC) and expression of fibrosis-related factors on HSC in vitro and further to explore the mechanism of Treg inducing fibrogenesis. MethodsHSC LX-2 cells were subcultured.CD4+ CD25+ CD127dim/- cells were purified using magnetic cell separation. The HSC were co-cultured with Treg by direct contact or by Transwell system in vitro. The HSC cultured alone was used as control. Cell proliferation was measured by CCK-8 assay.The expression of transforming growth factor (TGF)-β1 was detected by enzyme-linked inmunosorbent assay (ELISA), and the expressions of hyaluronic acid (HA) and precollagen Ⅲ (PC Ⅲ ) were detected by radio immunoassay (RIA). The data were analyzed by LSD-t test. ResultsHSC proliferation was strongest when Treg∶ HSC= 1.5∶ 1. The absorbance in direct contact co-culture group and Transwell system co-culture group was (0. 713±0. 032) cpm and (0. 735±0. 028) cpm, respectively, both of which were higher than that in control group [(0. 677 ± 0. 029) cpm](t = 5. 4003 and 8. 7878,respectively; both P＜0. 01). The concentrations of TGF-β1 in the supernatant were (781. 59 ±76.45) pg/mL and (813. 53±60. 62) pg/mL, respectively in direct contact co-culture group and Transwell system co-culture group, which were significantly higher than that in control group [(722.51±59. 66) pg/mL](t = 4.0014 and 6. 1653, respectively; both P＜0.01).The concentrations of HA were (433. 575±27.90) ng/mL and (445.40±23.73) ng/mL, respectively in direct contact co-culture group and Transwell system co-culture group, which were higher compared to that in control group [-(415. 83±19.44) ng/mL](t =3. 3124 and 5. 5231, respectively; both P＜0.01). Likewise, the concentrations of PCⅢ were (21. 93± 1.71) and (23. 125± 1.87) ng/mL in direct contact group and Transwell group, respectively compared to (20. 10± 1.49) ng/mL in control group (t = 4. 8082 and 7. 9436, respectively; both P ＜ 0.01).Furthermore, the absorbance,concentrations of TGF-β1, HA and PC Ⅲ in Transwell co-culture group were all higher compared to direct contact group (t = 3. 3875, 2.1639, 2. 2107 and 3.1354, respectively; all P＜0. 05).ConclusionsThe cell proliferation and the expressions of fibrosis-related factors in HSC increase greatly after co-cultured with CD4+ CD25+ CD127dim/-Treg. Therefore, Treg may play an important role in inducing liver fibrogenesis.

ObjectiveTo investigate the inhibitory effect of bone morphogenetic protein 7 (BMP7) on proliferation of rat hepatic stellate cells (HSC).MethodsHSC-T6 cells were cultured in vitro,and divided into four groups:control group,transforming growth factor (TGF) β1 group,TGFβ1 + BMP7 group,and TGFβ1 + BMP7 + Noggin group. The mRNA expressions of smad1,α-smooth muscle actin (α-SMA),gremlin and fibronectin (FN) were determined by semi-quantitative reverse transcriptasepolymerasechainreaction(RT-PCR).Theproteinexpressionsof phosphorylated-smad1/5/8 (P-smad1/5/8),α-SMA and gremlin were detected by Western blot.The statistical analysis were done using one-factor analysis of variance,LSD-t test,Dunnett's T3 test and Pearson linear correlation analysis.ResultsPhosphorylated-smad1/5/8,α-SMA,gremlin and FN were expressed on HSC-T6 cells in control group,which were significantly up-regulated after TGFβ1 stimulation (P＜0.05).The expressions of α-SMA,gremlin and FN were significantly down-regulated in TGFβ1 + BMP7 group compared with TGFβ1group, while P-smad1/5/8 expression was upregulated (1.613±0.031 vs.2.503±0.014; t=34.89,P＜0.05).The expressions of α-SMA,gremlin and FN in TGFβ1 + BMP7 + Noggin group were up-regulated than those in TGFβ1 + BMP7 group,while P-smad1/5/8 was down-regulated (t=34.05,P＜0.05).There was no difference of total smad1 mRNA expression among each group.ConclusionThese results collectively suggest that BMP7 strongly inhibits the expressions of α-SMA,gremlin and FN.

Objective To analyze the elonal gene rearrangement and complementarity determining region 3 (CDR3) Repertoire of TCR β-chain in fine needle aspiration biopsy (FNAB) specimens of peripheral T-cell lymphoma. Methods The TCR CDR3 region genes of 24 TCR Vβ subfamilies were amplified by utilizing RT-PCR technology, and the CDR3 size lengths of TCR β-chain were analyzed with genesean technology for 4 healthy individuals and 4 patients with peripheral T-cell lymphoma. The clonality of T cells presumed by spectratyping was further confirmed by CDR3 sequencing. Results TCR β-chain presented specific repertoire skewing in 4 cases with peripheral T-cell lymphoma, and only 1-4 TCR Vβ subfamily T cells were identified, respectively. Clonal expanded T cells, including mono, bioclonal and oligoclonal trend patterns, in one or more Vβ subfamilies were found in all cases. The mono expanded T cells have different CDR3 amino acid sequences. Conclusion Characteristic T cells cloning proliferation and selected usage of TCR Vβ subfamily T cells were found in 4 cases with peripheral T-cell lymphoma. The sequences of CDR3 in different TCR clone proliferation are different.