Objective To construct DNA vaccine expressing HIV-1 AE2f gp145-tat-rev-nef fusion gene( AE-Gp145TRN) and to compare the immunogenicities of DNA vaccines expressing Tat, Rev and Nef in gene fusion formulations of tat-rev-integrase(c-half)-vif-nef( AE-TRIVN) and AE-Gpl45TRN. Methods DNA vaccine was constructed by inserting the codon optimized HIV-1 AE2( gp145-tat-rev-nef fusion gene into mammalian expression DNA vector. In vitro expression efficiency of the constructed DNA vaccine was determined by Western blot and the immunogenicities of AE-Gpl45TRN and AE-TRIVN were compared by immunizing female BALB/c mice. IFN-r ELISPOT assay was used to read out the specific T cell immunity. Results Western blot assay showed the constructed DNA vaccine could be expressed efficiently in vitro. After vaccination, AE-TRIVN mounted significantly higher T cell responses against Tat, Rev and Nef[(148±91)SFCs/106 splenocytes]than Gpl45TRN[(55±28) SFCs/106 splenocytes]. Specific T cell responses elicited by AE-TRIVN predominantly targeting Rev, whereas Gpl45TRN could significantly enhance T cell responses against Nef. Conclusion AE-TRIVN and Gpl45TRN induced distinct T cell response modalities, which implied different gene fusion formulations may affect the immunogenicity of specific DNA vaccines.

In addition to the common complications involving hepatic artery, hepatic vein and biliary tract, which have already been mentioned and discussed in the preceding parts of this article, there are some uncommon complications which have been reported in the medical literature as the case report or as the case- series analysis. This paper sums up these uncommon complications. Part of these uncommon complications can be treated with interventional therapy. It is very important for interventional radiologists to make a further understanding of the different etiology of these uncommon complications occurred after liver transplantation so as to get a comprehensive knowledge about the complications after liver transplantation.

Objective To assess the osteogenic ability after co-culture BMSC and ADSC in vivo and in vitro.Methods ADSC and BMSC were obtained by adherent screening method and enzymatic digestion method.Flow cytometry was used to confirm the phenotypes of ADSC and BMSC.Oil red O was used to induce MSC to fat.Alkaline phosphatase (ALP) and alizarin red staining were used in osteogenic group.This sample was divided into four groups,no-induced stem cells group;BMSC osteogenic induction group;ADSC osteogenic induction group;co-culture of BMSC and ADSC osteogenic induction group.ALP activities and Calcium absorbance were determined during different periods of osteogenic introduction.OCN and Runx2 expression level were tested via RT-PCR and western blot methods after osteogenic induction for 2 weeks.Furthermore,cells in each group were seeded on HA/CS/PLLA composite scaffolds,and the scaffolds with cells were planted into bone defects in rat models.The rats were sacrificed by overdose anesthesia at 8 weeks after surgery and the scaffolds were removed for further analysis.Results Oil red O staining demonstrated red after adipogenic induction.Alkaline phosphatase and Alizarin red staining showed flaky red under condition of osteogenic induction.There had no statistical change among each group after osteogenic induction for 3 days,and ALP activity significantly increased after osteogenic induction for 5 days.Meanwhile,the ALP activity in co-culture of BMSC and ADSC group was markedly higher than the other three groups.However,there had no significant change in A value of calcium absorbance among each group after osteogenic induction for 7 days,while it increased at 14th day and ALP activity in co-culture of BMSC and ADSC group was significantly higher than the other three groups.After osteogenic induction for 2 weeks,the mRNA expression of OCN and Runx2 in co-culture of BMSC and ADSC group was 78.24±8.11 and 1 180.13±121.16 respectively,and the protein expression of OCN and Runx2 was 6.54±0.59 and 4.43±0.51.These mRNA and protein expression level in co-culture of BMSC and ADSC group enhanced significant compared with the other 3 groups.Histological assay demonstrated that the new bone tissues formed in co-culture of BMSC and ADSC group were 497.75±7.44 μm2,which was larger than that in the other 3 groups at 8 weeks after implantation.Conclusion Co-culture BMSC and ADSC may up-regulated the osteogenic ability in vivo and in vitro.

ObjectivePresent treatment in plastic surgery on giant congenital melanocytic nevus has always been a tough practice because it is difficult to achieve balance between effects and costs of treatment.This paper aimed to explore the concrete procedure of tangential excision and dermabrasion in treatment of adult giant congenital melanocytic nevus. Methods Taking into consideration pathological examination results before surgery,diseased regions,psychological expectancy and other factors,we used a humby knife or globe grinding head to remove giant congenital relanocytic nevus by wiping off the surface of it in 10 cases.After operation,the operated area of the skin underwent a process of healing in a moisturized state.In each case,surgical procedure was carried out by 1 2 sta ges,with the interval period ranges from 3 months to 6 months.ResultsOne to 3 years follow-ups showed that among those cases,5 cases obtained good results in which skin color of surgical area turned to normal and pathological examination showed that nevus cells disappeared,4 cases achieved improvement,and 1 case was relapsed.ConclusionsThe two alternative methods for treatment of giant congenital melanocytic nevus,either tangential excision or dermabrasion,with combination of pathological examination results,diseased regions,and psychological expectancy should be taken into consideration,which can remain a maximum balance between effects and costs of treatments.Tangential excision and dermabrasion are effective in some cases of giant congenital nevus where traditional methods do not work,or in order to reduce the cost of body appearance in treatment.Therefore,these two methods deserve to be adopted extensively in clinical therapy.But it still needs further accumulation of experience in practice and longer period of follow-up after operation.

Objective To explore the experience in diagnosis and treatment ot primary transitional cell carcinoma of prostate for the early and accurate to diagnosis and treatment.Methods The clinical data and features of 3 cases were retrospectively reviewed.Results All patients were diagnosed as primary transitional cell carcinoma of prostate.Two cases were advanced tumor.The preoperative examinations (ultrasound and serum,PSA) have failed to accurately indicate the diagnosis.All of them were confirmed by pathological examination.1 case lost follow-up,1 case performed TURP + chemotherapy through intravesical administration have survived 17 months respectively till today.The other case has already survived 2 months postoperatively but with lumbar spine bone metastasis.Conclusions Early diagnosis of primary transitional cell carcinoma of prostate is difficult.The diagnosis of the disease depends on the transrectal needle biopsy of the prostate or the specimens of the prostate after the Urethroscopy.Because of the prognosis is bad,and prone to pathological missed diagnosis or misdiagnosis.Need to exclude multicentric lesions and mixed tumor and choice of treatment method.

AIM: To investigate the anticancer effect of manumycin on abdominal metastatic breast cancer cell line-SK-BR-3 and its relationship with p38 MAPK . METHODS: The test of anticancer effect was performed by the method of MTT, apoptosis induced by manumycin and affected by SB203580, a specific p38 MAPK inhibitor, were examined by caspase-3 activity assay kit, and the protein expression was detected by immunoblotting assay. RESULTS: The inhibition rates at 24 h after treatment with manumycin of 6 ?mol/L, 18 ?mol/L, 54 ?mol/L were (7.4?3.9)%, (21.0?4.4)% and (64.7?4.1)%, respectively and showed dosage-effect relationship. Compared with the control group, the survival rates of the last two treatment groups were decreased significantly (P

Objective To explore the diagnostic value of CT angiography (CTA) of vertebral artery dissection (VAD) value. Methods We retrospectively analysed vertebral artery dissection in 30 patients (32 branches) according to the results of DSA. Tow radiologists independently analyzed the CTA images, and the sensitivity, specificity and accuracy of CTA in VAD patients were determined. The consistency of DSA and CTA results were evaluated by Kappa test. Results Thirty two branches of 60 vertebral arteries were diagnosed as VAD by DSA, 31 branches were diagnosed as VAD by CTA, 1 branch was misdiagnosis. Eight branches with dissection aneurysm were all displayed by CTA and DSA. Eleven branches of 12 branches withstring of beads signwere diagnosed by CTA. Five branches of 6 branches withstring and pearl signwere diagnosed by CTA;CTA and DSA of 1 branch withdouble-lumen signwere all displayed. Six branches of 5 branch withlinear signwere diagnosed by CTA. One branch showedlinear sign, but was diagnosed thrombosis by CTA. Two branches were showedlinear sign, but were diagnosedstring of beads signandstring and pearl sign. The sensitivity, specificity, accuracy of CT angiography in diagnosing VA dissection were 96.8％(31/32), 100％(28/28), 98.3％(59/60), respectively. The results made good agreement with DSA(Kappa=0.967,P<0.01). Conclusion Dual source CTA was a sensitive and accurate technique for the diagnosis of VAD.

BACKGROUND: It is important to study the methods of culturing bone marrow-derived mesenchymal stem cells (MSCs) to obtain a great amount of high purity MSCs for applying ocular tissues constructed by tissue engineering technique to treat eye diseases.OBJECTTVE: To separate and culture in vitro MSCs from bone marrow of the adult rats by density gradient centrifugation combined with adherence culture, and observe the growing characteristics and the possibility of mass multiplication.DESIGN: A completely randomized grouping design/repetitive measuring experiment.SETTING: Pathological laboratory, Zhongshan Ophthalmic Center, Sun Yat-sen University.MATERIALS: Four six-week-old SD rats about 250 g, grade Ⅱ of cleaning, were provided by the Animal Center of Sun Yat-sen University [certificate number: SCSK(Yue)2004/0011], about 250 g each rat and there was no limit to the sex. The main reagents and instruments included low sugar Dulbecco modified Eagle culture medium (DMEM/F12, American Gibco Corporation), trypsin (fetal bovine serum (FBS, Hangzhou Sijiqing Bio-Engineering Material Research Institute), American Gibco Corporation), disodium edetate, lymphocyte separating medium, fibronectin, CD44, CD34, CD31 monoclonal antibodies, two-step-method kit for immunohistochemistry (Beijing Zhong shan Biotechnology Corporation).METHODS: This experiment was conducted at the Key laboratory of Ophthalmology (Sun Yat-sen University), Ministry of ethanol (750 g/L) for 10 minutes. Under aseptic condition, the medullaris cavitas was exposed, the syringe containing application m edium was directly punctured into the femoral cavity, the cells in the medullaris cavitas were washed out with the culture medium containing heparin and taken as the cell suspension. The bone marrow-derived MSCs were separated and purified by density gradient centrifugation combined with adherence culture, and the growing conditions of the wells. When the cells had generally connected with each other, they were fixed with methanol or dimethy ketone in situ for 10 minutes, and then hematoxylin eosin (HE) staining or immunohistochemical staining. Antibodies against fiinoculated to 96-well culture plate by a cell density of 4.25×107/L, with 200 μL every well; then put the culture plate into culture box. Then from the next day to the sixth day, 5 g/L MTT solution was added into two rows (20 μL every well) every day, continuously cultured for 4 hours, then the supernatant was removed, and 200 μL DMSO was added to each well, agitated for 5 minutes, then detected the absorbance (A) values at 570 nm wave length, and a growth curve was drawn.branes were clear and the cell bodies were lucent. Being cultured for 2 days, there appeared adherent cells. 3 days later,most of the adherent cells extended and appeared to be in polygon, star or long-shuttle shapes. 4 days later, the cells showed to be in division growth stage; and about 12 days later, the cell clones were connected to each other, appearing curve of subcultured MSCs was in S shape. Cells began growing fast on the 2nd day after being passaged, and they entered the growth period for 3-4 days followed by saturation period, and then cells stopped growing.CONCLUSION: It is a simple and practical method to separate MSCs from the bone marrow of adult rats by means of density gradient centrifugation and adherence. MSCs can greatly proliferate in vitro and offer seed cells for the application of tissue engineering technique to treat eye diseases.

BACKGROUND: Bone sialoprotein (BSP) gene is expressed in human breast cancer cells, in which bone metastasis occurs easily outside the mineralized tissue. Clinical observation shows that the expression level of BSP of breast cancer cells at bone metastasis is higher that at the primary site;therefore, BSP may be closely related to tumor specific bone metastasis.The study on breast cancer bone metastasis can provide new drug target for clinical prevention and treatment.OBJECTIVE: To establish breast cancer cell strains of BSP with stable expression and observe the effect of BSP in the whole process of breast cancer bone metastasis.DESIGN: Controlled experiment.SETTING: College of Biological Sciences and Engineering, South China University of Science and Technology; Medical Experiment Center,Guangzhou General Hospital of Guangzhou Military Area Command of Chinese PLA.MATERIALS: This experiment was conducted in the Medical Experimental Center,Guangzhou General Hospital of Guangzhou Military Area Command of Chinese PLA,betweer November 2003 and March 2004..pIRES2-EGFP vector (5.3 kb) was purchased from BD Biosciences Clontech Inc.; E.Coli.Top10, pB-hBSP plasmid containing the coding region of hbsp, and human breast carcinoma cells, MDA-MB-231BR that was specifically transferred to brain and MDA -MB-231BO that was specifically transferred to bone.METHODS: hbsp gene was subcloned from pB-hBSP vector by PCR. Bg1Ⅱ and Pst Ⅰ restriction enzyme sites were inserted at 5' and 3' ends, orientation cloned to eukaryon expression vector pIRES2-EGFP, and constructed recombinant vector pIRES2-EGFP. The constructed recombinant vector was transfected into MDA-MB-231BR that was specifically transferred to brain and MDA-MB-231BO that was specifically transferred to bone.MAIN OUTCOME MEASURES: Construction of pIRES2-hBSP-EGFP recombinant expression vector; recombinant expression vector pIRES2-hBSP-EGFP transfecting breast cancer cells.Breast cancer strains specific in bone metastasis and brain metastasis were successfully transfected. The fluorescence labeling could be observed under the fluorescence microscope, and BSP had corresponding expression.CONCLUSION: The successful construction and transfection of pIRES2hBSP-EGFP of eukaryon expression vector would lay foundation for further study on the role of BSP in breast cancer metastasizing to bone in vivo or in vitro.

Objective To investigate the consumption rate of non-iodized salt,and evaluate the iodine status and goiter prevalence among school children in high water iodine areas of Henan Province from 2014 to 2015.Methods In the 20 counties with high water iodine,one township was randomly selected from each location (east,west,south,north and middle) in each county;secondly,4 villages were selected from each chosen township;thirdly,15 households were selected to collect salt samples from each chosen village.In the 10 chosen counties,one village with high water iodine was selected and water samples were collected;one school was sequentially selected from the chosen village and 100 school children aged 8-10 were chosen to collect their urine samples and measure their thyroid volume.Salt iodine was tested by semi-quantitative method;iodine contents of urine and water were tested by arsenic cerium catalytic spectrophometry;thyroid volume was measured by ultrasound method.Results In the 20 counties with high water iodine,4 440 salt samples were collected and tested both in 2014 and 2015;the rates of non-iodized salt were 98.2％ (4 363/4 440) in 2014 and 98.3％ (4 366/4 440) in 2015.In the 10 chosen counties,the median water iodine contents from the chosen villages were 202.0 μg/L in 2014 and 235.0 μg/L in 2015;970and 999 urine samples of the students from the chosen villages were collected and tested in 2014 and 2015,and the median urinary iodine contents were 251.9 μg/L in 2014 and 290.6 μg/L in 2015;937 and 948 students were examined in 2014 and 2015,the goiter rates were 3.4％ (32/937) in 2014 and 7.8％ (74/948) in 2015.Stratified by water iodine,the urinary iodine contents and goiter rates of school children increased with the rise of water iodine content.When the water iodine content exceeded 300 μg/L,goiter rate of school children was 8.4％,which was higher than other groups (P ＜ 0.05).Conclusions After stopping the supple of iodized salt in high water iodine areas,the current iodine status and goiter rate of school children are still higher than normal levels.Both noniodized salt supply and water improvement to reduce water iodine content should be taken in the areas with water iodine higher than 150 μg/L.