BACKGROUND:Stable and efficient labeling of dental pulp stem cel s in vitro is most important in tracer technique, which is also the basis of tooth regeneration in vivo.
OBJECTIVE:To determine the optimal condition and method for transfection of stem cel s derived from rat dental pulp with green fluorescent protein infection by lentiviral vector and to determine whether green fluorescent protein-labeled dental pulp stem cel s maintain their stem cel properties.
METHODS:Rat dental pulp stem cel s were obtained by modified enzyme digestion method, to identify the immune phenotype and differentiation potential. Dental pulp stem cel s were infected with green fluorescent protein by lentiviral vector for 24 and 48 hours at different multiplicity of infection (MOI) (5, 10, 25, 50 and 100). The infection efficiency and fluorescence intensity were analyzed by inverted fluorescent microscopy. The clonal and proliferation ability, cel cycle and the mineralization potential were compared before and after transfection. Based on those mentioned above, we could evaluate the influence of infection on their biological characteristics.
RESULTS AND CONCLUSION:Flow cytometry results showed that rat dental pulp stem cel s expressed STRO-1 and CD146 but not CD34 or CD45. The dental pulp stem cel s could differentiate into osteoblasts and
adipocytes when cultured in specific medium for each lineage differentiation. The highest efficiency of infection and strongest fluorescence expression appeared at 48 hours of infection and MOI 50. There were no significant differences in growth ability, cel colony formation rate and cel cycle before and after transfection (P>0.05). And the alkaline phosphatase expressed positively. Infection for 48 hours at MOI 50 is optimal for transfecting dental pulp stem cel s with green fluorescent protein by a lentiviral vector, thereby providing reliable tracer method for the study of rat dental pulp stem cel s in vivo.

Bone Transplantation ; Dental Research ; Humans ; Tooth

[ABSTRACT]AIM:Toestablishatransgenicheterozygousmousemodelofprecancerouslesionsofcolorectal cancer with p110δmutation in the C57BL/6J background for serving the studies on colorectal cancer research mediated by p110δ.METHODS:The transgenic heterozygous mice were generated by crossing in p110δD910A/D910A mouse and ApcMin/+mouse, and the genotype was detected by PCR .Compared with ApcMin/+mice, transgenic heterozygous mice ( ApcMin/+;p110δD910A/D910A)were counted, and the number and size of intestine polyps were analyzed after methylene blue staining . The intestinal tissue structure was assessed by HE staining .RESULTS:The transgenic heterozygous mouse model of pre-cancerous lesions of colorectal cancer with p 110δmutation was established .The number and size of polyps in the transgenic heterozygous mice were declined .CONCLUSION: A transgenic heterozygous mouse model of precancerous lesions of colorectal cancer with p 110δmutation was successfully established .The initial phenotype of intestinal tumors in transgenic mice was observed .This model will greatly contribute to the related research of colorectal cancer in mice .

Objective To explore the efficacy of multilevel anterior cervical discectomy and fusion for aged patients with cervical spondylotic myelopathy. Methods 25 aged patients with cervical spondylotic myelopathy were treated by multilevel anterior cervical discectomy and fusion. Japanese Orthopedic Association (JOA) scores were evaluated,clinical results were graded from excellent to poor using Odoms criteria,and the sagittal alignment of fusion segements were measured using Cobb method. Bone fusion and complications was evaluated. Results The final follow up score according to Odoms criteria was excellent in 8 patients ,good in 14 patients,and fair in 3 patients ,with excellent and good rate of 88%. The JOA score at final follow up(7.8) was significantly higher than preoperative(12. 8),with average improvement rate of 54%. At final follow up,bone fusion rate was 100%. Conclusion Multilevel anterior discectomy and fusion for aged patients with cervical spondylotic myelopathy was effective.

Objective To investigate the roles of sphingosine kinasel (SPK1) in apoptosis,invasiveness and multidrug resistance of human hepatocellular carcinoma cell line BEL-FU.Methods BEL-FU cells were infected with adenovirus carrying SPK1wT gene and SPK1siRNA (Ad-H1-SPK1) gene.Their effects on biological characteristics of BEL-FU cells were evaluated by MTT,cellular SPK enzyme activity assay,Transwell Migration Technology and Western-blot,respectively.Results AdSPK1wT significantly increased SPK activity but SPK1siRNA(Ad-H1-SPK1) decreased SPK activity.Over expression of SPK1 suppressed the apoptosis induced by DMS(Dimethyl sphingosine,DMS) and enhanced migration of BEL-FU cells.The cells infected with SPK1 siRNA( Ad-H1-SPK1)significantly increased the apoptosis induced by DMS and inhibited the migration of human hepatocellular carcinoma cells.The expression of multidrug resistance-related protein (MRP1) of cells infected with SPK1siRNA (Ad-H1-SPK1) was suppressed significantly compared with the control group,while the expression of MRP1 infected with Ad- SPK1wT was enhanced.Conclusion SPK1 activity is closely associated with apoptosis、migration and multidrug resistance of human hepatocellular carcinoma cells,therefore,it may serve as a new target for HCC treatment.

Objective To investigate the status of geriatric doctors' acknowledge and treatment of outpatients with lower urinary tract symptoms in 15 Chinese cities.Methods The investigation was conducted in 15 Chinese cities and involved 1056 geriatric doctors from December 2013 to February 2014.The questionnaire survey was made in behavior,attitude,skill and knowledge(BASK)of LUTS.The geriatric doctors participated in the investigation by answering the questionnaires about acknowledge for definition,etiology,diagnosis and treatment of the LUTS,and in results discussion,which was aimed at improving participants' cognition for diagnosis and treatment of elderly LUTS and at strengthening the practice.The final objective was to promote diagnosis standardization and to improve the level of clinical diagnosis and treatment for the elderly 1UTS.Results 95.9％ of geriatric doctors considered that they knew well the main clinical manifestations of LUTS.However,34.3 ％ of geriatric doctors were able to classify the symptoms of LUTS correctly.More than 90.0 ％ of geriatric doctors considered that the causes of LUTS were variable and they mastered the common cause of LUTS.But,38.0％ of them were able to completely master the causes of LUTS and 35.5％ would actively ask outpatients about the symptoms.70.0％ of the doctors thought they were familiar with the initial manifestations of LUTS and benign prostatic hypertrophy(BPH).However,many Chinese geriatric doctors didn't follow up the guidelines and 30.0％ of them failed to evaluate the severity of LUTS via IPSS(International Prostatic Symptom Score).75.8％ of geriatric doctors considered that they would give the patients the best treatment options by comprehensive assessment.However,60.0％ of them didn't know that α1 receptor blocker was the first line of treatment option of patients with moderate severe LUST.More than half of them were unclear that the patients with prostate volume under 40 ml could be treated by α1 receptor blocker.82.3％ of geriatric doctors considered that LUTS and hypertension should be treated respectively.But,55.0％ of them didn't know that ALLHAT trial has proved that Doxazosin would increase the risk of cardiovascular event.Conclusions The geriatric doctors are now able to pay attention to outpatients with LUTS,but still should further strengthen their acknowledge on the definition,cause,diagnosis and treatment of LUTS.

Objective To obtain alpaca single domain antibody targeting Her2.Methods An alpaca was immunized with human recombination Her2 protein mixed with Freund's adjuvant.Total RNA was extracted from the alpaca's blood and was used to synthesize first strand cDNA.Single domain antibody variable region (VHH) gene of the alpaca was amplified by PCR and cloned into pMES4 vector for library construction.After screening, E.coli BL21 (DE3) was transformed with selected clones and was induced with IPTG for the expression of recombinant proteins.The nanobody was purified by nickel ion affinity chromatography column.The affinity of the nanobodies to Her2 was tested.Results After the second round of screening, two antibody clones were selected, H3 and H5.The affinity of H5 was 8.106×10-10mol/L.Histochemistry results showed that H5 could recognize Her2 antigen in breast tumor tissue.Conclusion An Her2 specific nanobody derived from alpaca is obtained through phage display library screening, which can recognize human Her2 antibody in human breast tumor tissue.

Objective To investigate the relationship between prostate histologic inflammation classification and prostate cancer (PCa) in needle biopsy specimens with serum prostatic specific antigen (PSA) under 20 μg/L.Methods The clinical records of patients who underwent prostate biopsy were retrospectively analyzed in Beijing Hospital Urological Department from January 2011 to December 2013.The patients underwent prostate biopsy because of PSA raised and without pelvic radiotherapy.The needle biopsy specimen was evaluated by the pathohistologic criteria of location,extent and grade classification of prostate inflammation.Results PCa was detected in 84 (37.2％) biopsies and benign prostatic disease (BPD) in 142 (62.8％) biopsies.There were significant differences between PCa and BPD in inflammation classification (location:P＜0.001,extent:P＜0.001,grade:P＜0.001).On multivariable Logistic regression analysis,both location and extent classification of chronic histologic prostatitis were found tobe significantly associated with a lower risk of PCa in biopsy (location:P =0.001,OR =0.114,95％ CI 0.032-0.405 ; extent:P =0.021,OR =0.232,95％ CI 0.067-0.804).There was no relationship between grade classification and PCa (P=0.223,OR=1.805,95％CI 0.698-4.667).Under the evaluation of location and extent classification,it could differentiate PCa with BPD in biopsy with a sensitivity,specificity,positive predictive value and negative predictive value of 91.7％,50.7％,52.4％,91.1％,respectively.Conclusion The location and extent classification of chronic prostatitis is found to be associated with a lower risk of PCa independently.

cryopreservation periodontal tissue. METHODS:Periodontal tissue was scraped from healthy human teeth and divided them into three equal parts:fresh group, harvesting periodontal ligament stem cel s from fresh tissue;5%dimethyl sulfoxide group, 5%dimethyl sulfoxide added into the cryopreservation system;10%dimethyl sulfoxide group, 10%dimethyl sulfoxide added into the cryopreservation system. One month later, periodontal ligament stem cel s were extracted from the cryopreserved periodontal ligament from the latter two groups. RESULTS AND CONCLUSION:In the time that cel s swam out of tissue mass and cel harvest amount, the 5%dimethyl sulfoxide group was inferior to the fresh group but better than the 10%dimethyl sulfoxide group (P<0.05). No differences were found among the three groups in the fol owing aspects (P>0.05):colony formation rate of passage 1 periodontal ligament stem cel s, cel survival rate, proliferation ability of passage 3 periodontal ligament stem cel s, cel growth curve and surface marker expression of periodontal ligament stem cel s. The results suggest that the 5%dimethyl sulfoxide added cryopreserved system for periodontal ligament tissue cannot only shorten the time of periodontal ligament stem cel s amplification in vitro, ensure cel harvest and maintain basic cel ular biological characteristics, but also reduce the total amount of the dimethyl sulfoxide and the direct damage to the cel s caused by repeatedly frozen cel s, thereby providing a more secure guarantee for the future implementation of clinical transplantation therapy. So, the 5%dimethyl sulfoxide added cryopreserved system may be the new selection for donor tissue storage.

Objective To evaluate the etiology and clinical features of jejunoileum bleeding.Methods Seventy-two patients admitted in 7 hospitals of Shangdong province for jejunoileum bleeding from January 1998 to December 2008 were enrolled in the study. There were 46 males and 26 females with mean age of 47 years (ranged 13-85 years). The jejunoileum bleeding was confirmed by means of endoscopy, images or surgery. The causes, diagnostic methods and major clinical manifestations were retrospectively analyzed. Results The most frequent cause of jejunoileum bleeding was tumor (42/72,58.3 %), followed by enteritis (9/72, 12.5 %), diverticulum ( 7/72, 9. 7%), angiopathy (7/72,9.7%), Crohn's disease (3/72,4.2%). Differences were significant in constituent ratio of cause of jejunoileum hemorrhage between male and female and between jejunum and ileum (P＜0.05).Hematochezia or hematochezia with abdominal pain was the first presentation. The jejunoileum bleeding in 54. 2% patients was diagnosed by laparotomy, 23. 6% by capsule endoscopy, 9.7% by selective angiography, 6.9% by small bowel series and enteroclysis, 2.8% by colonoscopy and 2.8% by push enteroscopy. The complications of jejunoileum bleeding were anemia, intestinal obstruction,peritoneal metastasis, shock, ankylenteron and intestinal perforation. Conclusions Intestinal tumor is the most common cause in jejunoileum bleeding, especially in jejunum. Whereas the enteritis,diverticulum and angiopathy were often found in ileum. The capsule endoscopy and push enteroscopy are recommended in diagnosis of jejunoileum bleeding.