Objective To investigate the proliferation and collagen secretion of transplanted human fibroblasts.Methods The solution containing human fibroblasts(2?1010L-1)was prepared and 1 mL was injected into the dermis of BALB/CNU nude mice.Animals were killed by the end of the 1st,2nd and 3rd month after injection.The dermis in the injected area was taken out and stained with HE.Immunohistochemical staining for type I and type Ⅲ collagen was performed at the same time.Results Mitosis was observed by the end of the 1st,2nd and 3rd month.The concentration of type I and type Ⅲ collagen in the extra cellular matrix increased with the passing of time.Conclusion Transplanted human fibroblasts can proliferate automatically in the dermis of nude mice and manufacture the type I and type Ⅲ collagen in situ.Long period of survival and secretion will make it possible for fibroblasts to become promising option to correct minimal tissue defects.

ObjectiveTo analyze the distribution and density of Langerhans cells and dermal CD68 positive histiocytes in lesions of secondary keloid.MethodsTissue specimens were resected from the lesions of 30 patients with secondary keloid and normal skin of 14 human controls.Immunohistochemistry was performed to observe the expressions of CD68 and CD1a in these specimens.A micrometer was used to count the number of positively stained cells per unit area.The Student's t test was conducted for data analysis by using the SPSS software.ResultsThe density of CD1a+ Langerhans cells was (61 ± 49) cells/mm2 in the epidermis of secondary keloid lesions, (258 ± 61 ) cells/mm2 in the control epidermis,and(40 ± 65) cells/mm2 in the dermis of keloid lesions.CD68+ cells were absent in the epidermis of keloid lesions.Significant differences were observed in the density of CD1a+ Langerhans cells between the lesional and normal control epidermis(t =9.88,P ＜ 0.001 ) and in the percentage of CD68+ cells in nucleated cells between the superficial dermis of lesions and control skin(62％ ± 12％ vs.70％ ± 14％,t =2.66,P ＜ 0.05).The density of dermal CD68+ histiocytes was similar between the lesions and control skin ((287 ± 73) cells/mm2 vs.(290 ± 22) cell/mm2,t =0.02,P ＞ 0.05).Conclusions In keloid lesions,Langerhans cells decrease in the epidermis but increase in the dermis,CD68+ histiocytes are absent in the epidermis,and reduced in the dermis with a declined percentage in nucleated cells.

Objective To investigate the effect of small interfering RNA (siRNA) mediated silencing of △Np73 on 5-FU chemotherapy sensitivity in SW620 colocancer cells and provide new treatment approach for the colon cancer.Methods siRNAs were transfected into SW620 colon cancer cells.The expression of △Np73 was observed.Cell viability of colon cancer cells were measured by MTr assay and cell apoptosis was assessed with flow cytometry after treatment of control siRNA or △Np73 siRNA or combined with 5-FU,respectively.The tumorigenesis was assessed by injecting △Np73 siRNA or control siRNA transfeeted SW620 colon cancer cells into nude mice,followed by treatment with 5-FU in the tumors.Results △Np73 siRNA was able to strongly inhibit △Np73 expression,however,it did not inhibit the growth of cells.Combination treatment with △Np73 siRNA and 5-FU produced significant higher apoptotic cell(42.9%) as compared with those with 5-FU treatment(18.9%) alone or those with △Np73 siRNA(8.8%) alone.The treatment with 5-FU in the xenngrafts derived from △Np73 siRNA transfected SW620 cells in nude mice can inhibitor tumor growth significantly (t=15.32,P<0.05).Conclusion △Np73siRNAs can specifically repress the expression of △Np73.Thus it sensitizess the cells to 5-FU chemotherapy in colon cancer.

Objective The aim of this study was to establish a reasonable protocol for a rat model of ferric chloride-induced carotid arterial thrombosis by ultrasonic continuous evaluation.Methods Twenty SD rats were randomly divided into four groups and then treated with 20％,30％,40％ or 50％ concentrations of FeCl3,respectively.The vascular conditions were evaluated by 14L ultrasonic probe,at 10 mins,15 mins,and 20 mins.We then selected the best group through the examination of the rate of spontaneous reperfusion of blood vessels and the rate of reperfusion after intravenous injection of urokinase.At the end of the experiment,vessels were fixed in 10％ formalin solution and stained with HE.Results After external application of FeCl3 on rat common carotid artery for 20 mins,the artery occlusion rate was 100％,20％,0％ and 0％ in animals receiving 50％,40％,30％ and 20％ FeCl3,respectively.After external application of FeCl3 on rat common carotid artery for 120 mins,the spontaneous revascularization rate was 0％ in 50％ concentration group whereas were 100％ in rest other groups (P＜ 0.001).In 50％ concentration group,the partial recanalization rate was 40％ after intravenous injection of urokinase.HE staining revealed that the thrombus was dense and the lumen was partially recanalized after the urokinase intervention in 50％ concentration group.Conclusion By use of uultrasonic continuous evaluation of ferric chloride-induced thrombosis of rat common carotid artery,we have demonstrated that external application of 50％ ferric chloride solution for 20 mins is effective for the formation of thrombus model,which may be suitable for the studv of thrombolysis.

Objective To explore an anatomical basis for the lateral tarsal artery pedicle flap on front and lateral compartment of leg and the feasibility of repairing skin defects on forepart of feet. Methods The branches, course and anastomosis of the lateral tarsal artery, perforator of peroneal artery up external malleolus, superficial peroneal artery were studied in 20 legs of adult cadavers.The flap was designed on these grounds. 8 cases repaired by lateral tarsal artery pedicle flap on front and lateral compartment of leg, 5 cases of skin defects on dorsum of foots, 3 cases of skin defects on footplates.The area of defect on forepart of foot was 5 cm× 4 cm-cm × 5 cm. The donor sites were resurfaced with skin grafts or sutured directly. The lateral tarsal artery, perforator of peroneal artery up external malleolus, perforator of anterior tibial artery superficial peroneal artery were anastomosed each other, formed single band blood vessel axle on lateral foot, fore external malleolus, front and lateral compartment of leg. The area of flap was 6 cm × 4 cm - 10 cm × 6 cm.Results All of the flaps survived completely. All cases were followed up, followed up 6- 12 months, averaged 8 months. The color, appearance and texture of the flaps were good, without ulcer on the flap. The patients can walk freely. Conclusion The flap on front and lateral compartment of leg should be designed according to the lateral tarsal artery. Blood supply of flap was reliable, little trauma. The flap's vessel pedicle is enough long. It could repair any defect on forepart of foots.

Spotted fever caused by spotted fever group rickettsiae (SFGR) is found throughout China. During 2007—2008, 28 human SFGR isolates and 34 rat SFGR isolates including 15 isolates from Rattus fulvescens, 5 isolates from R.edwardsi, 7 isolates from Callosciurus erythraeus roberti and 7 isolates from Dremomys rufigenis) were obtained from L929 cell culture. Previous research indicated that the 62 strains of SFGR mentioned above shared not only the same serophenotype but also 100% of identity sequences of 16S rRNA, gltA, ompA, groEL and 17KD, which enabled us to apply multispacer typing (MST) to the 62 SFGR isolates in the study. Six primer pairs, which were used for typing of Rickettsia rickettsii and Rickettsia conorii, were chosen, and the results exhibited greater nucleotide polymorphisms among the 62 isolates tested. A total of 48 distinct genotypes were identified. The dominant genotype, represented by h3 isolates, accounted for 21.7% (13/60) of the isolates tested, and the remaining 47 genotypes were all unique. Phylogenetic analysis showed that all the 48 genotypes could be classified in the same clade, while the genetically related strain, R.heilongjiangensis, was close but not the same as the cluster. We concluded that the genetically diverse of spotted fever group rickettsiae strains are endemic in Chengmai County, Hainan Province, China.

Objective To explore the relationship between hypermethylation and expression of cadherin 1(CDH1)with lung cancer.Methods The semi-quantitative real-time methylation specific polymerage chsin reaction(MSP)were used to detect the promoter's relative methylation ratio of CDH1 in all 30 cases of lung cancer tissues,para-cancer lung tissues,distal lung tissues and 5 non-cancer lung tissue samples.Nested RT-PCR was used to detect the expression ratio of CDH1 mRNA.The western blot analysis was used to detect the expression of E-cad protein.The immunohistoeheroical method was used to confirm some negative results of western blot analysis.Results The relative methylation ratios was 0.13%-450.67%(median:33.61%)in cancer tissues,0.00%-177.02%(median:18.04%)in para-cancer tissues,and 0.00%-51.68%(median:13.69%)in far-cancer tissues.Statistical significance between cancer and pars-cancer tissues(Z=-2.355,P＜0.05)and between cancer and disial tissues(Z=-3.527,P＜0.01)were found.The results of nested RT-PCR showed that there were statistical sigaificance of mRNA expression between lung cancer tissues and pars-cancer tissues,distal lung tissues or non-cancer lung tissues(F=9.081,P＜0.01).The results of western blot showed that the positive expression rate of E-cad was 36.7% in lung cancer tissues,70.0%in para-cancer tissues,and 96.7% in far-cancer lung tissues,respectively.There was a statistical significance of positive rate pf E-cad expression between lung cancer tissues and pars-cancer tissues or diatal tissues(X2=6.70,24.30,7.68,P＜0.01).Conclusions The study showed that transcription of CDH1 mRNA would be silenced by hypermethylation of CDH1 promoter,resulting in the decreased expression of E-cad.The aberrant hypermethylation of E-cad is implicated in lung cancer.

Spotted fever caused by spotted fever group rickettsiae (SFGR) is found throughout China. During 2007–2008, 28 human SFGR isolates and 34 rat SFGR isolates including 15 isolates from Rattus fulvescens, 5 isolates from R. edwardsi, 7 isolates from Callosciurus erythraeus roberti and 7 isolates from Dremomys rufigenis) were obtained from L929 cell culture. Previous research indicated that the 62 strains of SFGR mentioned above shared not only the same serophenotype but also 100% of identity sequences of 16S rRNA, gltA, ompA, groEL and 17KD, which enabled us to apply multispacer typing (MST) to the 62 SFGR isolates in the study. Six primer pairs, which were used for typing of Rickettsia rickettsii and Rickettsia conorii, were chosen, and the results exhibited greater nucleotide polymorphisms among the 62 isolates tested. A total of 48 distinct genotypes were identified. The dominant genotype, represented by h3 isolates, accounted for 21.7% (13/60) of the isolates tested, and the remaining 47 genotypes were all unique. Phylogenetic analysis showed that all the 48 genotypes could be classified in the same clade, while the genetically related strain, R. heilongjiangensis, was close but not the same as the cluster. We concluded that the genetically diverse of spotted fever group rickettsiae strains are endemic in Chengmai County, Hainan Province, China.

Objective To establish a single-tube detecting system for the simultaneous identification of JAK2 V617F and JAK2 exon12 mutations.Methods Genomic DNA of cell line PC-3 was utilized as the wild type control,while genomic DNA of cell line HEL and plasmids with diverse JAK2 exon 12 mutations were used as the positive controls for JAK2 V617F and exon12 mutations.Multiplex PCR was performed to amplify the different amplicons combined with high-resolution melting (HRM) analysis,which established the multiplex detecting system for JAK2 V617F and exon12 mutations.Meanwhile 42 cases of polycythemia vera patients were collected to detect 2 kinds of JAK2 mutations by the above system and routine methods.Results The multiplex JAK2 mutations detecting system was successfully established by multiplex PCR combined with high-resolution melting curve analysis,which could simultaneously detect JAK2 V617F and JAK2 exon12 mutations.The analytical sensitivities of 2 mutations in this system were both up to 5％ and the precision (coefficient of variation) of intra-and inter-assay of the melting temperature (Tm) of 2 amplicons were separately less than 0.01％.37 cases were identified JAK2 V617F mutations from 42 polycythemia vera patients,while 2 JAK2 exon12 mutations cases were found from 5 JAK2 V617F negative patients.Compared with routine methods,the results matched the rate of 100％.Two cases of JAK2 exon 12 mutations were confirmed to the mutation types of H538K539delinsL and F537-I546dul10 + F547L by cloning and sequencing.Conclusions This method can simultaneously detect two kinds of JAK2 mutations in the peripheral blood and will contribute to the molecular diagnosis of myeloproliferative neoplasms,especially polycythemia vera.

Objective T o detect the genotype of A B O blood group by SN aPshot technology. Methods D N A w ere extracted from the peripheral blood sam ples w ith know n blood groups (obtained by serology) of 107 unrelated individuals in Y unnan. Six SN P loci of the 261th, 297th, 681th, 703th, 802th, and 803th nucleotide positions w ere detected by SN aPshot M ultiplex kit, and relevant genetics param eters w ere cal-culated. Results In 107 blood sam ples, the allele frequencies of types A , B , O A, and O G w ere 0.3551, 0.1682, 0.2300 and 0.2476, respectively, w hile that of types A G and cis A B w ere not detected. T he geno-typing results of A B O blood group w ere consistent w ith that of serologic testing. Conclusion SN aPshot technology can be adapted for genotyping of A B O blood group.