Objective To analyse the cloning and sequencing of mature fragment of human bone morphogenetic protein-4 gene. Methods The template DNA was extract from the human osteosarcoma cells line U-2OS by the single-step isolation method with isothiocyanic acid guanidine, the cDNA coding for the mature fragment of BMP-4 was amplified by the reverse transcription-polymerase chain reaction(RT-PCR). The mature fragment of BMP-4 was cloned into the vector pUC19, and sequenced by Sanger dideoxy-mediated chain termination method. Results The mature fragment of BMP-4 cDNA was obtained by RT-PCR and identified by sequencing. The computer search was done on Genebank, and the published DNA sequence of BMP-4 from Genebank (D30751) was chosen for a reference. Analysis showed that the homology and similarity of nucleotides and amino acids between cDNA of rhBMP-4 mature fragment and the published sequence of BMP-4 were both 99％ . Sequence analysis revealed that there were two bases mutations, one was at base 1 154(201) G C, this had no influence on the corresponding amino acids(Val), another was at base 1 222(269) C T, the mutation at the base 1 222 had turned the Ala into Val. Conclusion The mature fragment of BMP-4 gene has been cloned. The gene is of great significance in treatment of skeletal injuries and diseases.

ObjectiveTo study the cloning and sequencing of mature fragment of human bone morphogenetic protein-4 gene. Methods The template DNA was obtained from the human osteosarcoma cell line U2OS. By using RT- PCR method, the cDNA coding for the mature fragment of BMP-4 was amplified, cloned into the vector pUC19, and sequenced by Sanger Dideoxy-mediated Chain Termination method. Results The mature fragment of BMP4 cDNA was obtained by RT-PCR and determined by sequencing. Through the computer search on Genebank, the analysis showed that the homology of nucleotides and amino acids between cDNA of rhBMP4 mature fragment of this study and the published sequence was 99％. Sequence analysis showed that there were two differences, one was at base 1154 (201): G→C, which had no influence on the corresponding amino acids (Val). Another was at basel222 (269):C→T, the mutation at the base 1222 had the change of Ala to Val. Conclusion The mature fragment of BMP4 gene has been cloned. The results will be of great significance in treatment of skeletal injuries and diseases.

Objective To investigate the biological characteristics , therapy selection of tubular breast carcinoma.Relationship between axillary lymph node metastasis and other factors are analyzed retrospectively . Methods Tubular carcinoma patients were collected in our hospital from June ,1987 to March,2014.Paraffin tis-sues were re-checked again by pathology doctors and all patients were performed follow -up by telephone to e-valuate the prognosis .Results Sixty eight cases of tubular carcinoma of the breast were chosen .Axillary lymph node metastasis was closely related to tumor size and tumor grade in our research .In all patients ,only one patient was relapsed ,24 patients died of other diseases ,without distant metastasis or death of breast cancer .Conclusion Tubular breast carcinoma has a good prognosis with low recurrence rate .The treatment of tubular carcinoma is not quite reasonable .Preoperative core needle biopsy and surgery axillary staging should be paid more attention and breast conserving rate is still needed for improvement .

Objective To investigate the compliableness and cardiac rehabilitation effect of application of the Android‐based cardiac rehabilitation risk assessment simulative software in the patients with coronary heart disease (CHD) .Methods A total of 150 discharge patients with CHD treated in the cardiology department of multiple hospitals in Guangzhou City from December 2014 to December 2015 were selected as the research subjects and divided into the observation group for applying the Android‐based car‐diac rehabilitation risk assessment software and control group according to the random number table ,75 cases in each group .The compliableness and cardiac rehabilitation effect after this software out‐of‐hospital application in the CHD patients were observed . Results Among 150 cases ,141 cases continued to follow up ,the follow up rate was 94 .0% ,including 72 cases in the observation group and 69 cases in the control group ,the re‐hospitalization rate and the standard‐reaching rate of blood pressure ,blood glucose and blood lipid in the observation group were superior to those in the control group with statistical difference (P<0 .05);the com‐pliableness of behaviors following doctor′s advice and comprehensive evaluation of living quality in the observation group were su‐perior to those in the control group ,and the differences were statistically significant (P<0 .05) .Conclusion The application of the Android‐based cardiac rehabilitation risk assessment software is an effective measure for out‐of‐hospital cardiac rehabilitation , which can increase the compliableness of cardiac rehabilitation in the CHD patients .

Objective To investigate the status of living relative kidney transplantation, and the clinical effects and social significances of kidney transplantation between spouses. Methods We retrospectively collected the clinical data of the department of kidney transplant of the First Affiliated Hospital of Zhengzhou University, from January 2011 to December 2013. The spouse group as group 1, the age and sex of doners were taken into account,the siblings with the similar age of the same period were enrolled in group 2. Then the postoperative recoveries of the two groups were compared. Considering the current social status, particularly the shortage of donor kidneys, the clinical, social and family significances of kidney transplantation between spouses were analyzed. Results Twelve cases of spouses in group 1, 8 cases of siblings in group 2 , the differences of donor and recipient age of the two groups were 0.33 ± 0.98 years and 2.29 ± 7.23 years, respectively. The human major histocompatibility complex antigens (HLA) was less than three in group 1, and was greater than or equal to three in group 2. The changes of serum creatinine and urea nitrogen were analyzed. No significant differences of serum creatinine and downward trend of blood urea nitrogen were observed between two groups (P = 0.84, P = 0.79). Conclusion The kidney transplantation between spouses has good clinical efficacy and great social significance, improving the status of the shortage of donor kidney and contributing to family harmony. The renal transplantation between spouses has obvious advantages and need further promotion.

Objective To study the local irritation of repeated intrathecal injection of Ziconotide Acetate,and to provide reference for irritancy evaluation ofintrathecal injection.Methods Sixteen New Zealand rabbits were assigned into two groupsat random:Control group and Ziconotide Acetate group,eight animals each group.Totally 50 μL saline or Ziconotide Acetate (100 μg/mL) were administrated by repeated lumbar intrathecal injection once daily for 7 d.Animal behavior was observed every day,and four animals in each group were sacrificed 2 d later after the last injection,the lumbar spinal cord was removed for histopathological examination and irritancy evaluation.The remaining animals were sacrificed for initancy evaluation 14 d later after the last injection.Results Only one animal died after anesthesia on day three in saline group,while no obvious adverse reactions were observed in other rabbits during the entire study,and no intrathecal irritant reactions of histopathological examination were found in both groups.The reversible minor mechanical damage was observed at the injection point,2 d after the last administration.Conclusion For 7 d repeated lumbar intrathecal injection in rabbits,no intrathecal irritant reactions observed in Ziconotide group,and the New Zealand rabbit could be used as a local irritation evaluation model.

ObjectiveTo express the hepatitis C virus (HCV) core gene in E . coli on a high level. Methods　The cDNA coding for HCV core protein was amplified by polymerase chain reaction (PCR). The PCR product was　purified and digested with restriction enzymes and inserted into the downstream of PRPL promoter of a high-level ex- pression vector pBV220. HCV core gene was expressed in E. coli in a non-fused form. The expression protein was analysed by SDS-PAGE , and its immunoactivity was tested by ELISA. Results Sequence analysis of the amplified PCR products confirmed that we have successfully cloned and expresssed the intact core protein of HCV. SDS-PAGE showed that a specific protein with a molecular weight of 21kDa at a level of 14. 0％ of the total bacterial proteins ap- peared in bacteria harboring pBV/HCVCore, while this protein was absent in the control bacteria harboring pBV220. The results of enzyme immunoassay analysis showed that this protein could be specifically recognized by the HCV pos- itive sera from patients with hepatitis C . Conclusion The intact HCV core protein was successfully expressed in E . coli in a non-fused form on a high level, and its immunoactivity was high.

Objective To dynamically monitor the changes of peripheral blood lymphocyte subsets of renal transplant recipients and investigate the relationship between lymphocyte subsets with infection and rejection.Methods The clinical data of allogenic kidney transplantation recipients and living relative donors in the Department of Kidney Transplantation of the First Affiliated Hospital of Zhengzhou University were prospectively collected from June 2015 to December 2016.The data of lymphocyte subsets and other related indexes were obtained from renal transplant recipients and relatives of the same period.Results Sixty-four cases of living-relative donors and 351 cases of renal transplant recipients were enrolled in this study,and the recipients were divided into 3 groups:infection group (67 cases),acute rejection group (46 cases),and stable group (238 cases),according to the diagnostic criteria.There was significant difference in the concentration distribution of lymphocytes between the stable group and the control group (P<0.05).The stable frequency distribution range of the stable group was as follows (cells/μL):Lym (1 000-1 500),T (<1 500),CD4+ (<1 000),CD8+ (<1 000),B (<300),NK (100-300),CD4+/CD8+ (0.5-1.0).The number of Lym,T,CD4+,CD8+,NK and B cells in the preoperative patients was less than that in the healthy population (P<0.05);The number of Lym,T,CD4+, CD8+,B and NK cells was gradually decreased in the postoperative infection group,which was less than that in the stable group (P<0.05).After treatment the indicators gradually restored to the level in the stable group level;the number of T,CD4+,CD8+,B cells was highly correlated with infection.The number of T and CD4+ cells,and CD4+/CD8+ ratio were significantly increased in acute rejection group as compared with the stable group,and gradually decreased after the rejection was reversed.The number of T,CD4+,CD8+ cells was highly correlated with rejection.Lymphocyte subsets had a predictive effect on infection and rejection of recipients,and CD4+ cell count and CD4+/CD8+ ratio were independent risk factors.Conclusion The monitoring of lymphocyte subsets has an important clinical value in the evaluation of immune status and individual treatment of renal recipients.

Objective To evaluate the characteristics and efficacy of microscope-assisted bilateral decompression via unilateral approach for the treatment of lumbar stenosis. Methods From June 2007 to June 2010, Sixty case lumbar stenosis with bilateral decompression were treated via unilateral approach under microscopy. Patients were followed up from 6 to 24 months, average (12 ± 4.7) months. Results The pain level of each patient was assessed both before and after the opeartion, using a visual analogue scale (VAS). Intermittent claudication was completely relieved in 57 out of 60 cases, moderately relieved in 3 cases. VAS score decreased from pre-operational 9.08 ± 0.76 to post-operational 2.33 ± 1.43, and there was significantly difference between them. There was no recurrent case during the whole follow-up. Conclusion Bilateral decompression via unilateral approach under microscope is proved to be an effective and safe procedure for the treatment of lumbar stenosis, and have the advantages of minimal invasion, less pain, quick recovery, better effect, little influence on the spinal stability.

Purpose To prepare 68Ga-NOTA-SPIO as PET/MRI dual mode imaging probe with high sensitivity and resolution,and further evaluate its in vitro and partly in vivo biological properties.Materials and Methods The precursor SPIO-PEG2000-NOTA was prepared and characterized.The precursor was radiolabeled by using one step method to prepare 68Ga-NOTA-SPIO as dual mode imaging probe.The labeling rate of the probe was determined by rapid thin-layer chromatography.Besides,the in vitro stability and lipid water partition coefficient of the probe were evaluated,and its biodistribution in normal mice was also observed.Results The precursor SPIO-PEG2000-NOTA with uniform dispersion and uniform particle size was prepared,and the dual mode probe 68Ga-NOTA-SPIO was synthesized.The labeling rate reached 99％,and the lipid water partition coefficient (Log P) was (-2.60±0.13).The radiochemical purity of the probe was higher than 95％,as it was incubated in the phosphate buffer and fetal bovine serum within 2 hours.The probe was mainly distributed in the liver and spleen of mice,and its clearance velocity in blood was fast.Conclusion The double mode probe 68Ga-NOTA-SPIO synthesized by one step method has high labeling rate with no need of purification,which has good physic-chemical properties and biocompatibility.The probe can be used in the further research of PET/MRI dual modality imaging.