Objective To investigate the immunological activity and the effect of an anti－ keratin monoclonal antibody 5G5 on the proliferation of serum－ free cultured keratinocytes. Methods The reactivity of 5G5 with cultured keratinocytes was observed by immunohistohemistry technique. The keratins extracted from the cultured keratinocytes was identified by SDS－ PAGE and their reactivities with 5G5 were assessed by immunoblot analysis. MTT was used to study the influences of 5G5 on the proliferation of cultured keratinocytes. Results 5G5 recognized the band of keratin with molecular weight of 50 000 only. Positive staining of 5G5 was found in the cytoplasm of cultured keratinocytes. The strong potential of 5G5 to inhibit the proliferation of cultured keratinocytes was shown by MTT assay with dose－ dependent manner. Conclusions The antibody which specifically recognizes the keratin with molecular weight of 50 000 may be the effective component to inhibit the proliferation of cultured keratinocytes.

Purpose:To study the effect of insulin-like growth factor 1 receptor ?-strain(IGF1R-?) interrupted by a special antibody (IGFⅠR-?Mab)on lung adenoma cell line SPC-A-1.Methods:IGFⅠR-?Mab was extracted by hybrid technology. SPC-A-1 cells were separated into 2 groups,the IGFⅠR-?Mab and the blank control. The IGFⅠR-?Mab cells were interfered by different densities of IGFⅠR-?Mab, including 20,40,60,80,100,120,140,160,180 and 200 ng/ml. The MTT curve line, morphology, ultrastructure and cell cycles were observed at 0,24,48,72 hours after the intervention respectively. Results:Compared with the control, apoptosis in IGFⅠR-?Mab group was significant(P= 0.009)and proliferation rate was decreased obviously within 160 ng/ml. However, the proliferation rate was no significant when the special antibody density was more than 200 ng/ml.Conclusions:The affinity of IGFⅠR-?Mab at IGF1R ?-strain is high. The interruption of IGF1R ?-strain by IGFⅠR-?Mab shows the obvious biological effects in vitro ,with inclusion of promoting apoptosis and suppressing proliferation, which indicate the interruption targeting IGF1R ?-strain is prospective for non-small-cell lung carcinoma.

Objective To identify hardly recognizable victims of an accident. Methods Tissues of muscle and cartilage were obtained from the dead bodies. Some of the tissues were checked by routine pathological microscopy. Genomic DNA was isolated from the tissues and subjected to STR profiling of 16 sites via multiple fluorescent PCR analysis with ABI’s AmpFLSTR Amplification Kit. Individual identification of the victims was carries out by matching the STR profiles of the victims with those of the parents. Results Routine pathological microscopy showed that the structure of some of the muscle tissues was totally destroyed, while the structure of all cartilage tissues was basically intact. Three patterns of genomic DNA isolated from victims’ muscle tissues could be seen in gel electrophoresis, i.e. basically undegraded, partially degraded and totally degraded. STR profiling failed due to the degradation of genomic DNA of some of the muscle tissues, while all samples of the cartilage genomic DNA could be used for STR typing. Conclusion Paternity identification based on STR genotyping was an effective way to identify victims of accidents, and cartilage tissue from the victims was the first choice for that purpose.

ObjectiveTo establish a analytical system for the survival motor neuron (SMN) subtle mutation,and evaluate its application in two families with spinal muscular atrophy (SMA).MethodsSMN genes in seven family members from two SMA families were analyzed at both transcript level and genomic level,by the use of the conventional PCR-RFLP,allele-specific PCR,multiplex ligation-dependent probe amplification (MLPA) and T subcloning and sequencing of SMNI gene.ResultsIn family A,the patient had a single SMN1 copy who was carrying nonsense mutation L228X,which was also found in his father.In family B,as the patient's sample was unavailable,the father was indeed a carrier with one normal SMN1 allele and the other SMN1 allele carrying a frameshift mutation 22_23insA.The remaining family members were SMA carriers with one SMN1 copy.ConclusionThis analytical system for SMN subtle mutation offers viable molecular basis for genetic counseling and prenatal diagnosis in SMA families.

Objective To obtain recombinant human pancreatic alpha-amylase protein (AMY2A). Methods Human pancreatic AMY2A cDNA was synthesized by RT-PCR using total RNA from human pancreatic tissues and a couple of primers designed according to the know sequence of human pancreatic alpha-amy lase gene, then digested with BamH Ⅰ and Kpn Ⅰ and inserted into the prokaryotic expression plasmid pGEX-5T vector. Construct The prokaryotic expression vector pGEX-5T-AMY2A was constructed and transformed into E. coli BL21 cell . Protein expressed under the induction of IPTG. The inclusion bodies were isolated and solubilized with urea and washed denatured and refolded. The fusion protein wes purified by affinity chrom atography with glutathione agarose. Results Sequence and restrction analysis revealed AMY2A gene was cloned in frame into pGEX-5T, SDS-PAGE profile showed a clear protein band with a relative molecular weight of 84000 and western blot indicated that the expressed product specifically reacted to polyclonal anti -human pancreatic AMY2A genes. Conclusion Human pancreatic AMY2A gene was successfully cloned,expressed and purification.

Objective To investigate molecular diagnostic method for hereditarymethemoblobinemia. Methods The cDNA coding sequence of NADH-cytochrome b5 reductase (b5R) from 3 patients with hereditary methemoglobinemia was analyzed by direct sequencing of RT-PCR products and the genomic DNA of b5R gene by PCR-restriction endonuclease digestion or PCR-sequencing. Results The b5R cDNA of patient A was T/C heterozygous at nucleotide 527 and G/A heterozygous at nucleotide 608. The b5R cDNA of patient B was G/A heterozygous at both nucleotide 170 and nucleotide 179. The b5R cDNA of patient C was G/A heterozygous at nucleotide 608 and C/T heterozygous at nucleotide 791. Result of genomic DNA analysis was in agreement with that of cDNA approach. Conclusion The method for molecular diagnosis of hereditary methemoglobinemia was established and 3 novel b5R gene mutations were identified in compound heterozygosity in 3 Chinese patients.

Objective To construct a recombinant vector of sperm-specific human lactate dehydrogenase ( hLDH-C4 ), express it in Escherichia coli ( E. coli ) BL21 ( DE3 ) and utilize it in the detection of anti-sperm antibody. Methods The coding sequence of hLDH-C4 was amplified from human testis λTripIEx cDNA library, and inserted into pET-28a( + ) after restriction enzyme digestion with Hind Ⅲ and Xho Ⅰ. The resultant recombinant vector was used to transform E. coli BL21 ( DE3 ) and the His-Tag fused hLDH-C4 was expressed after induction with IPTG. Western blot was used to analyzed the recombinant protein and LDH activity of bacterial lysates was determined. An indirect ELISA method for the detection of anti-sperm antibody was established by using the recombinant hLDH-C4 as antigen matrix. Results pET-28a( + )-hLDHC was successfully established. The protein with size of 35kD could be induced by IPTG when the recombinant plasmid was transfected into E. coli BL21 ( DE3 ). Western blot showed that the recombinant protein could be specifically recognized beth by anti-His tag monoclonal antibody and by rabbit anti-human LDH-C4 antibody. In addition, the recombinant protein showed high-level LDH activity when the bacterial lysate after IPIG induction was used to check LDH activity. The recombinant hLDH-C4 was confirmed when it was used in indirect EL1SA to detect anti-hLDH-C4 antibody. Conclusions The coding sequence of hLDH-C4 is cloned into the vector pET-28a( + ) and recombinant hLDH-C4 was expressed at a high level in E. coli. The recombinant hLDH-C4 is useful in the detection of anti-sperm antibody.

Objective To investigate the clinical effect and safety of kidney transplantation from donors with severe hand-foot-mouth disease (HFMD).Methods Five cases of kidney transplantation from three donors with HFMD between Jan.2014 and Dec.2016 were analyzed.The age of three donors was 2 years,2 years and one month,and 3 years and 11 months respectively,and body weight was 11 kg,10 kg and 15 kg respectively.The age of recipients ranged from 26 to 41 years and weight from 50 to 59 kg.Single kidney transplantations were performed on 4 cases,and dual separating kidney transplantation on one case.Results One case of the transplantations was failure due to the allograft artery thrombosis.The rest 4 cases gained satisfied clinical effect.None of the 5 cases showed any symptoms associated with HFMD.Conclusion The clinical effect of kidney transplantation from donors with severe HFMD is satisfactory.The organs from donors with severe HFMD could only be used by adult recipients.

Objective To investigate methods for prenatal molecular diagnosis of fetuses at high risk for X-linked adrenoleukodystrophy(X-ALD).Methods The amniotic fluid was obtained and genomic DNA was isolated from amniotic fluid cells.Maternal contamination was evaluated by paternity test.PCRRFLP,sequencing and denaturing high performance liquid chromatography(DHPLC)were used to detect the ABCD1 gene of fetal genome.Results In the pedigree 1,the PCR product(799 bp)of the fetus 1 and her father(normal control)could be digested with BcnI. No P560L mutation,which was present in the index patient,was detected in the ABCD1 gene from the genomic DNA of the fetus 1 using direct sequencing.In the pedigree 2,the PCR product(232 bp)of the fetus 2 and her father could not be digested with MaeI and no Q177X mutation,which was present in the propositus,was detected in the ABCD1 gene from the genomic DNA of the fetus 2 using direct sequencing.In the pedigree 3,the PCR product(271 bp)was digested with AciI.the pattern of the fetus 3 and the propositus being the same,and the R617C mutation was found in the ABCD1 gene from the genomic DNA of the fetus 3 using direct sequencing.In the pedigree 4,the PCR product(269 bp)was analyzed with the DHPLC,and the pattern of elution peaks of the fetus 4 and her father was similar,but different from that of the propositus.No K276E mutation was detectable in the ABCD1 gene from the genomic DNA of the fetus 4 by using direct sequencing.Judging from the sex of the fetuses,fetuses 1 and 2 were normal homozygotes,fetus 3 was an ALD hemizygote,and fetus 4 was a normal hemizygote.Conclusion A new protocol for X-ALD prenatal molecular diagnosis is proposed,which would ensure the accuracy of prenatal diagnosis.

Parasitism characteristics of spirometra mansoni sparganum in the living donor kidney are analyzed by present cases and relevant literatures. A female aged 49 years voluntarily donated a kidney to her son. Results of healthy evaluation were accorded with the standards of living donor kidney. During repairing kidney, a sliver cyst was found in the adipose capsule on the kidney ventral surface, near to the renal hilum. The cyst was incised, and a ivory white girdle-shaped worm was obtained. After identification, the worm was identified spirometra mansoni sparganum (living body). Pathological examination showed that the cyst developed granulomatous inflammation, combined with neutrophil and eosinophilic granuiocyte infitration. Following surgery, the donor and recipient were treated with praziquantel. No proglottid or worm ovum was detected by dung detection within 3 months, without any discomfortable symptom. The infection mode and pathway may be by eating unmatured paratenic host meat or infected cyclops. The donor and recipient should be examined for parasitic infection of sparganosis mansoni prior to transplantation. No significant symptom could be detected following parasitism of sparganosis mansoni in the kidney, so it was seldom found. Worm ovum was examined in feces, which could be the evidence for sparganosis mansoni and for case history inquisition. Eosinophilia in the blood always indicated that chronic parasitic infection. Zoogenetic infection test could be tested when necessary. Sparganum antigen could be used for various immunological tests, which could provide evidence for auxiliary diagnosis of immunology. The diagnosis was usually confirmed by obtaining a polypide by surgery or histological examination. CT scanning and magnetic resonance imaging have diagnostic value of renal sparganosis mansoni.