Liver cirrhosis is a common chronic progressive liver disease,and at present the most effective treatment for advanced liver cirrhosis is liver transplantation.However,main reasons of limiting the wide application of liver cirrhosis are liver source deficiency,expensive cost,graft rejection reaction,the complications caused by long-term application of immunosuppressant and so on.Stem cell transplantation has become a new method for the treatment of liver diseases due to its beneficial to the damaged liver tissue repair,and it can compensate part of liver function.The basis and clinical research progress,the existing problems and prospects of the bone mesenchymal stem cell transplantation for the treatment of liver cirrhosis are summarized,aiming to provide theoretical basis for the further research.

Objective To investigate the effect of different promoters on the expression level of transgene containing MAR expression vector in recombinant CHO cells.Methods The CMV promoter and 3-globin MAR were amplified by PCR,then CMV promoter was replaced the SV40 promoter in pCAT1 for constructing the expression vector droved by CMV promoter.The control vectors of pCAT1 and pCAT2 without containing MAR were simultaneously transfected into the CHO cells.Then the stably transfected cell line was screened by G418.The CAT gene expression level was analyzed by ELISA.Results The expression level of CAT enzyme in the cells transfected with MAR-containing vectors was increased compared with the cells transfected by pCATG and pCAT3 vectors without containing MAR,which were increased by 1.75 and 1.25 times respectively(P＜0.05);but CAT enzyme expression level in the pCAT1 transfected cells droved by SV40 promotor with the MAR-containing expression vectors was 1.4 times higher than that in the pCAT2 vector droved by the CMV promoter(P＜0.05).Conclusion MAR can enhance the transgene expression level in stably recombinant CHO cells,and the promoting efficiency of SV40 promoter and MAR combination is superior to that of CMV promoter and MAR combination.

Objective To analyze the effect of GC-rich DNA fragments on the level of transgenic expression in Chinese hamster ovary (CHO) celts and its position effect.Methods The synthetic DNA fragment with GC-rich was cloned into the 5'or 3'or both 5'and 3'ends of expression cassette of expression vector.Three new expression vectors (pIRES-G1,pIRES-G2 and pIRES-G3) which was inserted with the GC-rich DNA fragments in different position were transfected CHO ceils,respectively,and then was observed under fluorescence microscope;the control vector was pIRES-EGFP.Stable transfected cell lines were screened under G418,and enhanced green fluorescent protein(EGFP) expression was analyzed by flow cytometry and the transgenic copy number was detected by quantitative real-time quantitative polymerase chain reaction (qRT-PCR).Results Three expression vectors with a GC-rich DNA fragments in different position were constructed successfully.The insertion of GC-rich DNA fragments at 3'end and both 5',3'ends of the box of expression vector could obviously improve the expression level of vector in CHO cells;and the expression level of the stably transfected CHO cells increased 1.39 fold and 1.32 fold compared to the control vector,respectively;the transgene copy number increased 1.32 fold and 1.24 fold compared with the control vector.While the insertion of GC-rich DNA fragments at 5'end of expression cassette had no obvious effect on the level of gene expression.Conclusion The role of DNA fragment with GC-rich in improving the transgenic expression of CHO cells is related to its position in the vector.The insertion of GC-rich DNA fragments at 3'end and both 5',3'ends of the box of expression vector can improve transgenic expression.

Objective To investigate the expression of hsa-miR-144 in esophageal squamous cell carcinoma, and its relationship with clinicopathological features and prognosis. Methods Reverse transcriptase polymerase chain reaction (RT-PCR) method was used to detect the hsa-miR-144 in 46 cases of esophageal squamous cell carcinoma and adjacent normal tissue. The expression of hsa-miR-144 in esophageal squamous cell carcinoma and its difference in the clinicopatho?logical characteristics including gender, age, and tumor size were investigated. The relationship between the expression of hsa-miR-144 and prognosis of patients with esophageal squamous cell carcinoma was analyzed. Kaplan-Meier method and Log-rank test were used to analyse the differences in survival rates in different pathological characteristics. Results The ex?pression level of hsa-miR-144 was lower in esophageal squamous cell carcinoma 0.97(0.22-24.48)×10-6 than that of adjacent normal tissue 8.60(0.09-258.20)×10-6, the difference was statistically significant (Z=2.221, P<0.05). The expression level of hsa-miR-144 was higher in esophageal squamous cell carcinoma with no lymph node metastasis than that in esophageal squa?mous cell carcinoma with lymph node metastasis (Z=2.758,P<0.05), and the expression level decreased with the increase in the pathological staging (Z=7.737,P<0.05). There were no significant differences in the expression levels of hsa-miR-144 between different gender, age, tumor size, tumor location, tumor differentiation and tumor invasion depth (all P>0.05). There was no correlation between the expression of hsa-miR-144 and prognosis in patients with esophageal squamous cell carcino?ma (rs=0.031, P=0.839). In the survival rate, there was no statistic significance between high expressive of hsa-miR-144 group and low expressive group (P=0.828). The survival rate was lower in patients with lymph node metastasis than that of pa?tients without lymph node metastasis. The survival rates were lower in patients with relatively deep invasion and higher patho?logic stage (P<0.05). Conclusion The expression of hsa-miR-144 is down regulated in esophageal squamous cell carcino?ma, and which is associated with lymph node metastasis and pathological staging of esophageal carcinoma. It shows that hsa-miR-144 may serve as an anti-oncogene in the occurrence and development of esophageal squamous cell carcinoma.

Objective To evaluate the anti-HBV effect of hypericin from the cellular level and to preliminarily explore its po-tential drug target point.Methods Liver cell line HepG2.2.15 cells secreting HBV particles were selected as the experimental ob-jects.Hypericin served as the HY group,lamivudine was taken as 3TC group and deionized water as the blank control group.The cells were grouped and administrated.The HBV-DNA copy level was measured at72 h after medication by Southern blot and fluo-rescent quantitative PCR;the inhibition rate of HBsAg and HBeAg was detected by using ELISA assay;the pgRNA expression level was tested by using Northern blot and fluorescent quantitative PCR;Western blot and fluorescent quantitative PCR were adopted to detect the expression of regulatory factors including HNF3β,HNF4α,PPARαand RXRα.Results Compared to the blank control group,both hypericin and lamivudine had significant inhibiting effect on HBV DNA and expression level of HBsAg and HBeAg in HepG2.2.15 cells (P <0.05).Hypericin could significantly decrease the pgRNA expression compared with the blank control group (P <0.05),while lamivudine had no obvious change (P <0.05).Moreover,hypericin exhibited significant effects on the expression of HNF3βand regulatory factor HNF4αcompared with the blank control group and 3TC group(P <0.05).Conclusion Hypericin represents a strong anti-HBV effect,moreover could increase the negative regulatory factor HNF3βn expression and decreases the positive factor HNF4αexpression,prompting that its drug target point could be pgRNA.

Objective To investigate the relationship between the plasticity of dendritic spines in entorhinal cortical neurons and mechanism of low-dose ketamine-induced reduction of cognitive dysfunction following sevoflurane anesthesia in aged rats.Methods Thirty-six pathogen-free healthy male SpragueDawley rats,aged 18 months,weighing 500-600 g,were divided into 3 groups (n=12 each) using a random number table:control group (group C),sevoflurane anesthesia group (group Sev) and ketamine group (group K).Group C received no treatment.Group Sev inhaled the mixture of air (flow rate 1 L/min) and 3.6％ sevoflurane for 3 h.In group K,ketamine 10 mg/kg was injected intraperitoneally,and 5 min later the mixture of air (flow rate 1 L/min) and 3.6％ sevoflurane was inhaled for 3 h.Open field test and Morris water maze test were performed 3 days after anesthesia.After the behavioral tests,the animals were sacrificed,and their brains were removed and cut into sections for determination of the density of neurons,density of dendritic spines,and expression of postsynaptic density protein-95 (PSD-95) and synaptophysin (SY38) in superficial laminaes (Ⅱ-Ⅲ) of entorhinal cortex using Nissl's staining,Golgi staining and immunohistochemistry,respectively.Results Compared with group C,the time of staying at the central region was significantly shortened,the escape latency was prolonged,the density of dendritic spines was decreased,and the expression of PSD-95 and SY38 was down-regulated in group Sev (P＜0.05).Compared with group Sev,the time of staying at the central region was significantly prolonged,the escape latency was shortened,the density of dendritic spines was increased,and the expression of PSD-95 and SY38 was upregulated in group K (P＜0.05).There were no significant differences in the density of neurons in entorhinal cortex between the three groups (P＞0.05).Conclusion The mechanism by which low-dose ketamine attenuates cognitive dysfunction induced by sevoflurane anesthesia may be related to the enhanced plasticity of dendritic spines in entorhinal cortical neurons of aged rats.

Carcinoma, Squamous Cell ; metabolism ; mortality ; Esophageal Neoplasms ; metabolism ; mortality ; Humans ; MicroRNAs ; metabolism ; Prognosis

Objective To evaluate the value of human epididymis secretory protein 4(HE4)and CAl25 in the diagnosis of ovariall malignancy.Methods HF4 and CA125 in the serum specimens of malignant ovarian tumor group(30 cases),benign ovarian diseases(110 cases;45 benign ovarian tumor,57endometriotic diseases and 8 pelvic inflammation were included) and healthy women group( 137 cases)were assayed double blindly . The levels and the diagnosis efficiency of the HE4 and CA125 were analyzed.Results (1) The median levels of HE4 and CA125 were significantly higher in malignant ovarian tumor group (244 pmoi/L and 601 kU/L respectively) than those of the benign ovarian diseases group( 32 pmol/L and 22 kU/L respectively)and healthy women group (32 pmoi/L and 11 kU/L respectively) (P =0. 000-0. 029). The median levels of CA125 were also higher in endometriotic diseases and pelvic inflammation groups(53 and 41 kU/L respectively) than those of benign ovarian tumor group and healthy women group (12 and 11 kU/L respectively;P = 0. 000-0. 031 ). (2) The positive rate of HE4 was lower than that of CA12s in malignant ovarian tumor group ( P = 0. 036 ). HE4 was negative in benign diseases and healthy women groups. But the positive rates of CA125 were 56. 1% and 5/8 respectively in endometriotic diseases and pelvic inflammation groups and there were significant differences compared with HE4( P =0. 000). (3)The HE4 assay had advantage over the CA125 assay in receiver operating characteristic-area under the curve (ROC-AUC) and sensitivity with a specificity of 100% when ovarian malignancy was compared with controls having benign diseases and healthy women, benign tumor or benign diseases groups respectively. The CA125 assay had advantage over the HE4 assay in ROC-AUC and sensitivity with the same specificity when ovarian cancers were compared with controls having healthy women group. (4) Combined assay of HE4 and CA125was better than CA125 alone when ovarian malignancy was compared with controls having any group. (5)Combined assay was better than HE4 alone in ROC-AUC and sensitivity with the same specificity when ovarian cancers were compared with controls having benign diseases and healthy women or healthy women groups. And combined assay was lower in the ROC-AUC and the sensitivity with specificity of 100% than HE4 when ovarian cancers were compared with controls having benign tumors or benign diseases groups respectively. (6) The diagnosis efficiency of the HE4 assay at the level 86 pmol/L determined in ROC curve with controls having benign diseases and healthy women group and at the 95% reference level 50 pmol/L of healthy women or 150 pmol/L recommended by the kit respectively was compared. The sensitivity of 50 pmol/L was 73% higher than 150 pmol/L and 86 pmoi/L, while the specificity and positive predictive value were lower ( P = 0. 002, P = 0. 000 ). The specificity, accuracy and positive predictive value of HE4 assay at the set point of 150 pmol/L and 86 pmol/L were 100%, 96% and 96%. The set point of 86 pmol/L had advantage over 150 pmol/L at the sensitivity of diagnosis, 70% and 63% respectively. But the positive predictive value was 95% lower than 150 pmol/L, being 100%. There was no significant difference( P =0. 883, P = 0. 883 ). Conclusions The specificity of HF4 assay is higher than CA125 assay in the diagnosis of ovarian cancer and HE4 combined with CA125 assay can improve the diagnoses. The set point of 150 pmol/L is advantageous for the accurate diagnosis, while the set point of 86 pmol/L is advantageous for the screening of malignant ovarian cancer.

Among ankle injuries, the injury to the distal tibiofibular syndesmosis is common and likely neglected. The stability of the distal tibiofibular syndesmosis is related to the depth of the fibular notch. In imaging diagnosis, X-ray examination cannot be used for a definite diagnosis of the injury to the distal tibiofibular syndesmosis. For diagnosis of the distal tibiofibular separation>3 mm, CT scan can be accurate but is not sensitive enough for a separation<1 mm while MRI is more sensitive in diagnosis of the injury. Arthroscopy has gradually been used as the "gold standard" in diagnosis of the injury to the distal tibiofibular syndesmosis due to its advantage of direct vision. The distal tibiofibular separation occurs in the injuries of pronation external rotation Ⅳ°, supination external rotation Ⅲ° and Ⅳ°, and pronation abduction Ⅱ° and Ⅲ° by the Lange-Hansen classification. Most patients with simple stable injury to the distal tibiofibular syndesmosis may have a good prognosis after nonoperative treatment. Surgical anatomic reduction and maintenance of stability of the distal tibiofibular syndesmosis are the basic management principles for unstable distal tibiofibular syndesmosis or the injury to the distal tibiofibular syndesmosis combined with ankle fracture. Screw fixation is the most commonly used in the surgical treatment of the injury. Elastic fixation has the advantages of maintaining the biological characteristics of the distal tibiofibular syndesmosis fretting joint, better reduction fault tolerance, and lower rates of complications and long-term reduction loss. The angle of nail placement is the key to maintaining good reduction of the distal tibiofibular syndesmosis, but there has been little description of the specific methods to ensure the theoretical angle of nail placement in practice. This article also reviews the prospects of the future treatment of the injury to the distal tibiofibular syndesmosis.

Objective To screen out specific aldosterone(ALD)antibodies using phage display technology and recom-binant antibody technology,providing raw materials for the research and development of ALD diagnostic kits.Methods Five healthy and clean New Zealand white rabbits were selected and immunized for the first time against the diluted ALD-keyhole limpet hemocyanin antigen(2 mg·L-1)using a multi-point injection method on the back,with a dose of 1 mg per rabbit.Immunization was administered again every 2 weeks,with a 50％reduction in dose.Starting from the third immunization,the ear vein blood of the rabbits was collected one week after each immunization.A chemiluminescent plate coated with 0.25 mg·L-1 ALD-bovine serum albumin antigen was used to measure serum titers via indirect and competitive methods.After the 5th immunization,the rabbit with high serum titers and good specificity was selected,and its spleen and bone marrow were removed.The spleen tissue was grinded,and RNA was extracted using TRIzol reagent in one step to obtain gene sequences in the variable region of light chain(VL)and the variable region of heavy chain(VH).The single-chain variable fragment(ScFv)was connected through the linker and constructed into the bacteriophage vector Pcomb3xss;then,it was carried to Escherichia coli TG1 through electrotransformation,and the ALD ScFv phage display library was constructed accordingly.Three to five rounds of enrichment screening were performed against the library.Monoclonal clones,identified by enzyme-linked immunosorbent assay(ELISA)competitive method,were selected for phage supernatant preparation,and a highly competitive clone sequence was obtained.The screened clone sequence was inserted into the pCMV3 expression vector,and the HEK293 cell was transfected using the transient transfection method after the plasmid was extracted.One week later,the supernatant was collected,and its purity and expression were identified by affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).Results After the 5th immunization,the serum titers of 5 rabbits were indirectly tested,and the results showed that the serum titers of 4# and 5# white rabbits were still greater than 10,000 after being diluted by 32,000 times.The test results based on the competitive method showed that the ratio of low to high values in the plasma sample of 5#white rabbit was 2∶1,superior to that of other white rabbits.The 5# white rabbit was selected for phage library construction.The VL and VH gene fragments were amplified by conventional polymerase chain reaction,and then bridged into ScFv(VL+VH).The agar gel electrophoresis analysis showed that the size of the band was about 750 bp,which was consistent with the size of the originally designed fragment.ScFv was cleaved and electroporated into Escherichia coli TG1 to construct a phage library with a storage capacity of 4.73 × 108 cfu·mL-1.After 3 rounds of washing,300 monoclonal clones were selected from the outbound petri dishes to prepare monoclonal bacteriophages.The ELISA results showed a positive rate of 100％among the 300 clones,and 42 clones were tested positive for calibration competition,with a screening rate of 14％.The 42 positive clones were further subjected to clinical sample competition testing,and 16 monoclonal strains that met the requirements were screened.The 16 strains were retested,and the results of the two tests were consistent.After sequencing,6 antibody sequences were selected for construction and expression.After purification,SDS-PAGE reduced gel electrophoresis results showed that there were bands at positions 50,000 on the heavy chain and 25,000 on the light chain.Six highly affinitive and competitive rabbit ALD monoclonal antibodies were obtained.Conclusion Six highly affinitive and competitive rabbit ALD antibodies are successfully screened using phage display technology,which provides a reference method for the discovery of small molecule antibodies.The screened AD1 85 and AD277 antibodies show a competitive advantage twice that of the positive control in the competition of calibration and clinical samples,providing a possibility for the development of raw materials for ALD detection kits.