BACKGROUND: Human pulp tissue has been known to be less, and exhibit poor tolerance to enzymatic digestion and less adherent cells after step-by-step digestion of trypsin and collagenase, thereby often leading to a failure of passage. Only several kinds of dental pulp cells with poor activity can be obtained by the tissue explant-collagenase digestion. OBJECTIVE: To investigate human dental pulp cells cultured in vitro by tissue explant method. DESIGN, TIME AND SETTING: A cytological observation was performed at Heping Campus and School of Stomatology, Jilin University from 2005 to 2007. MATERIALS: Healthy young human teeth extracted for orthodontic correction or impaction. METHODS: Pulp tissue from the third molar teeth was collected, cut into small blocks with a size of 1.0 mm×1.0 mm×0.5 mm under the infiltration of small amount of Dulbecco's modified eagle's medium, and then transferred into a 6-well plate containing culture medium for incubation in a 5% CO2 and saturated humidity atmosphere at 37 ℃. During the process of incubation, pulp tissue was adjusted at a density of 3-6 blocks/well, with an equal spacing of 0.5 cm and the 6-well plate was kept inverted. Three hours later, the 6-well plate was turned over to make tissue blocks adhering to the plate wall. Culture was continued after addition of 2 mL of culture medium. Culture medium was renewed every 4-6 days. After 6-15 days, cells emigrated from the edge of tissue blocks and call outgrowth appeared around each tissue block. When cells closed to confluency, a digestion procedure of 2.0-3.0 minutes (0.25% trypsin and 0.02% ethylenadiamine tetraacetic acid) was followed by passage culture at a proportion of 1: (2-3) in 25 mL of culture flasks. Purified fibroblast-like cells were gradually obtained from primarily cultured cells by repeated digestion and passage. MAIN OUTCOME MEASURES: Cellular morphology was identified by immunohistochemistry; secreted dental pulp cells were determined using alkaline phosphatase activity; the growth curves of human pulp tissue cells were depicted by MTT assay. RESULTS: Under an inverted phase contrast microscope, the obtained dental pulp cells were primarily typical fibroblasts with a long-shuttled appearance, well-rounded call body, uniform cytoplasm, round or oval nucleus, and clear nucleolus. Immunohistochemistry results showed call surface vimentin-positive, pan cytokeratin-negative, and alkaline phosphatase-posltive These cells were decreased after culturing 1 day, were slightly increased after 2 days, entered the logarithmic growth period and were markedly increased after 4 days, entered a platform period after 8 days, and began to decrease again after 9 days. The whole growth curve of cells appeared in "S" shape.CONCLUSION: The dental pulp cells isolated from human pulp tissue by tissue explant method can effectively proliferate end retain a poody differentiated state in vitro.

Objective To analyze the residents′ willingness to hierarchical medical system and their intention of first visit hospitals.Methods Customized questionnaire was used for in-home survey of 1 500 households in three cities of Shandong province,with 773 of the residents aware of hierarchical medical system used as the study samples.Descriptive analysis and multi factor Logistic regression were employed for statistical analysis of their intention.Results 85.0% the residents expressed willingness to embrace hierarchical medical system.55.0% of the residents prefer primary medical institutions as their first visit in case of mild illness,a choice chosen by more rural residents(58.9%)than urban residents(48.0%),a difference of statistical significance(P<0.01).In the case of severe illness,87.3% of them prefer major hospitals as their first visit,and more urban residents(94.3%)take this option than the rural residents(83.3%),a difference of statistical significance(P<0.01).Service ability of medical institutions and residents′ concept for medical service played a key role in their choice of first visit hospitals.Conclusions The key to ensuring the implementation effect of hierarchical medical system lies in better service ability of primary medical institutions and correction of residents′ irrational medication concepts.

Objective:To establish a method based on the iPLEX analysis of MassARRAY mass spectrometry platform to detect multiplex genetic mutations among Chinese lung cancer patients. Methods:We reviewed the related literature and data of lung cancer treatments. We also determined 99 mutation hot spots in 13 target genes, namely, EGFR, KRAS, ALK, FGFR1, FGFR2, FGFR3, PIK3CA, BRAF, PTEN, MET, ERBB2, AKT1, and STK11, which are closely related to the pathogenesis, drug resistance, and metastasis of lung cancer and are associated with relevant transduction pathways. A total of 297 primers comprising 99 paired forward and reverse amplification primers and 99 matched extension primers were designed by using Assay Design in accordance with the mutation label and format requirements of the MassARRAY platform. The detection method was established by analyzing eight cell lines and six lung cancer specimens;the proposed method was then validated through comparisons with a LungCarta kit. The sensitivity and specificity of the proposed method were evaluated by directly sequencing EGFR and KRAS genes in 100 lung cancer cases. Results:The proposed method could detect multiplex genetic mutations in the lung cancer cell lines, and this finding is consistent with that observed using previously reported methods. The proposed method could also detect such mutations in clinical lung cancer specimens;this result is also consistent with that observed by using the LungCarta kit. However, an FGFR2 mutation was detected only by using the proposed method. The measured sensitivity and specificity were 100%and 96.3%, respectively. Conclusion:The proposed MassARRAY technology-based method could detect multiplex genetic mutations among Chinese lung cancer patients. Indeed, the proposed method can be potentially applied to detect mutations in cancer cells.

Objective To analyze the correlation factors that promote or impede the residents to preferentially use essential medicines.Methods Adopting stratified random sampling method,1 700 households selected from 5 cities of Shandong province were investigated with a questionnaire.The framework of Andersen behavior model of health service utilization was used as the framework,with such methods as descriptive analysis and univariate logistic regression models for the analysis and evaluation of relevant information.Results The residents′ tendency factor,ability factor and environmental factor influence their preference to use essential medicines,while the requirement factor plays a minimal role.There was a significant difference for the preference of combined medication,first visit preference and self-medication experience,the efficacy and policy response of essential medicine from logistic regression analysis.Conclusion At present,the government should focus on the construction of the formation mechanism of the residents′drug use behavior and the policy response mechanism of essential medicine system.

50%;failure,defined as no remission or no deterioration of symptoms;and overall response rate, defined as cure plus improvement. Results The overall response rate was 98% (45/46), and the complete cure rate was 87% (40/46).The mean operative time in patients undergoing bladder neck suspension [(66.7?9.8)min] was significantly longer than those undergoing mid-urethral suspension [(35.1?12.1)min] (P0.005).Shortly after operation,5 cases of bladder neck suspension (28%) and 3 cases of mid-urethral suspension (11%) had urine retention (P

Objective To assess the safety,efficacy and reliability of IUPU(Abbreviation of Institute of Urology,Peking University)technique in setting up the retroperitoneal cavity for retroperitoneoscopy. Methods From February 1996 to March 2006,more than 1100 retroperitoneoscopic procedures were performed with the IUPU technique in setting up the retroperitoneal cavity,First,at the cross point of the line 2 cm above the iliac crest and the longitudirml line close to the anterior auxiliary line,a 1 cm skin incision was made and the Veress needle was penetrated into the retroperitoneal space(RPS)with a 0°-30° angle to the perpendicular line.The CO2 gas was pumped into the RPS till the pressure increases to 14mmHg and the first port was inserted into the RPS.The laparoscope was then inserted into the RPS through the first port and kept swinging right and left with its tip and trunk tO set up the retroperitoneal cavity.Other 2 ports were put into the RPS at cross points of the sub-costal line and anterior and posterior auxiliary lines under monitor observation.Then other appliances are introduced into the RPS to expand the cavity.More than 1100 procedures had been done with the IUPU technique,including 54 cases of simple nephrectomy (loss of function due to tuberculosis,hydronephrosis and atrophy), 188 radical nephrectomy, 154 ureteronephrectomy, 344 adrenalectomy, 302 renal cyst decortications, 35 partial nephrectomy, 37 pyeloplasty. Results The average time for the IUPU technique was (5.4±2.8)min (range 4. 5 to 14. 5 min) to set up the RPS. Complications included converting to open surgery due to bleeding in 8 cases(8/1114,0. 72%) when the first port was inserted into the RPS and entered into the peritoneal cavity for the first port penetration in 32 cases (32/1114,2. 87%),although the retroperitoneal cavity could be set up successfully by adjusting the laparoscope into the RPS.There was no injury to other viscera. Conclusions The IUPU technique is safe, efficient and reliable in setting up the RPS and no other special instrument is needed. It can be finished within 5 min on skilled hands and is valuable as a routine method to set up the retroperitoneal cavity.

Objective To establish a HRM assay to screen for KRAS mutations in clinical colorectal cancer patients.Methods The sensitivity of HRM was analyzed by detecting somatic mutations in exon 2,notably codons 12 and 13 of the KRAS gene in the serial plasmid mixture samples which were mixed using the different proportions mutation plasmid and wide type plasmid of KRAS.HRM analysis was performed for KRAS on DNA insolated from a panel of 60 colorectal cancer samples derived from fresh tissues.The results were compared with the direct sequencing data.Results After the PCR amplification,the mutation results could be available by performing HRM analysis in the same tube on a real time PCR machine with HRM capability.HRM detection could identify KRAS mutation in a proportion of 10% of mutation plasmid DNA.All 60 samples identified the KRAS mutation by HRM and sequencing.17 samples were positive(28.3%) by HRM for KRAS exon 2 mutations,and 15 samples were confirmed the presence of codon 12 or 13 mutations(25.0%) and the other 2 samples were wild type by sequencing.The 60 samples detected by HRM were given 100% sensitivity with 96% specificity.Conclusions HRM is a sensitive intube methodology to screen for mutations in clinical samples.HRM will enable high-throughput screening to gene mutations to allow appropriate therapeutic choices for patients and accelerate research aimed at identifying novel mutations in human cancer.

ObjectiveTo investigate the fusion sequence complexity of EML4-ALK in non-small cell lung cancer (NSCLC) patients,and the potential mutation in tyrosine kinase ( TK ) domain of ALK gene.MethodsIn routine practice,a novel echinoderm microtubule-associated protein-like4 and anaplastic lymphoma kinase (EML4-ALK) V3c variant was detected by rapid amplification of cDNA ends-polymerase chain reaction ( RACE-PCR )-sequencing technology in a patient with NSCLC.The further consecutive 39 cases( total of 40 cases)were screened by use of reverse transcription (RT)-PCR for EML4-ALK fusion.Positive PCR products were purified and cloned into T vectors,transformed into DH5a germ cells and colony picked up and sequenced for sequence complexity analysis.Tyrosine kinase domain of ALK was amplified by RT-PCR and sequenced.ResultsThree out of 40 cases had EML4-ALK fusion.One case had six novel variants of EML4-ALK co-existing,termed as V3c ( 64.6％ ),V3d ( 25.0％ ),V3e ( 2.1％ ),V3f (4.2％ ),V3g(2.1％ )and V3h(2.1％ ) variants,whereas without common V3a and V3b variants.In other two positive cases,one was V1 variant,another was concurrent V2,V3a and V3b variants.No mutations were detected in the TK domain of EML4-ALK in any case.ConclusionsSeveral EML-ALK variants could co-exist in a given lung cancer tissue,which suggest that the diversity and sequence complexity of EML4-ALK fusion are exist.Attentions should be paid to screen all the variants in clinic to improve the pick-up rate.

Objective To evaluate the safety and efficacy of the anatomical retroperitoneoscopic nephrectomy(RSN)and standardize the procedure of RSN. Methods The retrospective analysis was performed on 405 consecutive patients underwent anatomical RSN in Our institute from January 2002 to June 2008.There were 232 male and 173 female patients with the average age of(57.2±14.2)years,among whom there were 228 renal cell carcinoma patients accepted RSU,96 and 49 renal pelvic carcinoma and ureteral carcinoma cases accepted retroperitoneoscopic ureteronephrectomy (RSUN) and 32 cases accepted simple RSN due to loss of renal function caused by benign renal discsses.The tadical RSN was performed by dissecting outside Gerota's fascia and in the latent cavities between this fascia and lateral conal fascia in the dorsal side and between this fascia and prerenal fusion fascia in the ventral side,whereas the simple RSN was done inside Gerota's fascia by making direct incision on it and dissecting between this fascia and perirenal adipose tissue.Kidneys and perirenal adipose tissue were completely removed by dissection along several avascular planes around the kidney under the amplified view of laparoscopy. The software SPSS 12.0 was used for the statistical analysis of all data. Results The mean operative time was (132±48)min for radical and simple RSN and (245 ± 62)min for radical RSUN, which included the time for position change and second skin preparation. The medium estimated blood loss was 100 ml(10-2500 ml) and the average drainage volume was 150 ml (0-1152 ml) postoperatively. 15 cases (3. 70%) required blood transfusion with the median volume of 400ml (400-1650 ml). Four cases (0. 99%) were converted to open surgery due to severe adhesion (2 cases), difficult exposure of renal helium (1 case) and severe bleeding (1 case).The mean drainage time was (3. 9±1.8)d, the mean time to first oral intake was (2.7±1.2)d and the mean postoperative hospital stay was (8.6±3. 8)d. Conclusion The anatomical RSN is safe and effective and should be the standard surgical procedure for laparoscopic nephrectomy.

Background and objective All the advanced NSCLC patients that received EGFR-TKI therapy will eventually relapse after a period of efficacy. The aim of this study is to investigate the serum biomarkers as potential predictive factors for the efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) targeted therapy in advanced non-small cell lung cancer. Methods Twenty serf-paired serum samples were collected from 9 advanced NSCLC patients that evaluated as disease control (SD or PR) after gefinitib therapy, at the time points of before and after gefinitib treatment but 2 weeks before being evaluated as disease progress. AII samples were pre-separated by WCX microbeads, and then detected on the MALDI-TOF-MS platform of Bruker AutoflexTM. ClinProTools (Version: 2.1) was used to analyze the differentially expressed proteins. Results There were 7 protein peaks (m/z), 3 242.09, 8 690.36, 2 952.64, 3 224.04, 1 450.51, 1 887.8 and 3 935.73 found statistically differentially expressed between the self-paired samples. Three proteins (3 242.09, 2 952.64 and 3 224.04) were down-regulated and four proteins (8 690.36, 1 450.51, 1 887.8 and 3 935.73) up-regulated in gefinitib treated sera. Conclusion The data here suggest that several specific protein peaks might indicate gefinitib resistance, yet the identities of these proteins and the mechanisms underlying the responsiveness to gefinitib treatment need further investigation.