Objective To construct recobinant pmRNA IRES-hKDR and lay a primary foundation for further study of gene therapy for tumors.Methods pmRNA IRES-hKDR was obtained from pDC520 hkDR and pmRNA IRES-Luc which was reserved by our laboratory through molecular biology technology and was transfected into Hepall-6 via lipsome after identification of PCR,restriction enzyme digesting and DNA ELISA and Western blot.Results The recombiant plasmid had pmRNA IRES-hKD gene which was expressed in Hepall-6//G418 in vitro and the expressed protein was secreted out of the cells could response with specific hKDR antibody,which was identified by Western blot.Conclusion An eukaryotic expression recombiant plasmid pmRNA IRES-hKDR can be constructed successfully which provides the possibility of further researches on its anti-tumor effect.

Objective To investigate the immune protection of dendritic cells(DCs) harboring kinase domain-containing receptor(KDR) fusion gene on mice carrying B16 melanoma.Methods The bone marrow precursor-derived dendritic cells(BMDCs) were induced from bone marrow progenitors of mice by GM-CSF.The KDR fusion gene mRNA was transfected into DCs in vitro.Mice were immunized with the same amount of DCs at 7-day interval and then each mouse was injected with 5?10~5 B16 cells.The mice without tumors were injected with B16 cells again 20 days later.Mice were randomly divided into 4 groups: group A(n=7),group B(n=8),group C(n=8) and group D(n=7).The mice were immunized with DCs one time in group A,2 times in group B,3 times in group C and as controls in group D.Results After one week,all mice in group D had tumors with average survival 15 days.All mice in group A,B and C had no tumors after 20 days later.After the second injection of B16 cells,2 mice in group A and 2 mice in group B had tumors.The mice in group C had no tumor.The average survival periods were calculated from first injection of B16 cells to the study end.The average survival period of group A was 50 days and that of group B was 72 days.Conclusions The dendritic cells vaccine harboring KDR fusion gene has immune protection against melanoma in mice.

Objective To assess the value of bulbocavernosus reflex (BCR) in the differential diagnosis of Parkinson' s disease (PD) and multiple system atrophy (MSA).Methods BCR was compared in 55 patients with PD,20 patients with MSA,and 50 healthy controls,who were enrolled from the Department of Neurology,the First Affiliated Hospital of Wenzhou Medical University from May 2013 to April 2014.The clinical features of autonomic nerves dysfunction were also recorded.Results Among all related autonomic symptoms,the occurrence rate of constipation,urinary urgency and frequency in patients with MSA was higher than those with PD.The elicit percent of BCR in patients with PD was 93％,higher than those with MSA (70％).The average latency of BCR in patients with MSA was longer than those with PD (tmale left =16.275,tmale right =14.269,tfemale left =5.954,tfemmale right =5.905,all P ＜ 0.05).The degree of BCR amplitude decreasing in three groups was MSA ＞ PD ＞ healthy controls.There was statistically significant difference among three groups (Fmale left =75.73,Fmale right =73.13,Ffemale left =72.70,Ffemale right =59.44,all P ＜ 0.05).The area under receiver operating characteristic curve (ROC) in differential diagnosis of PD and MSA of the average latency of BCR in male and female was 0.947 and 0.948.The area under ROC curve in differential diagnosis of PD and MSA of the average amplitude of BCR in male and female was 0.886 and 0.920.The ROC curve showed the average latency of BCR in male of 44.80 ms with a sensitivity of 95％ and a specificity of 84％,and in female of 61.35 ms with a sensitivity of 86％ and a specificity of 88％ ; the average amplitude of BCR in male of 0.37 mV with a sensitivity of 96％ and a specificity of 68％,and in female of 0.36 mV with a sensitivity of 98％ and a specificity of 76％,which were critical cutoff values in differential diagnosis of PD and MSA with the best sensitivity and specificity.Conclusion The latency and amplitude of BCR test helps to increase the accuracy in the differential diagnosis of PD and MSA.

BACKGROUND:Synovial mesenchymal stem cells have the ability of multilineage differentiation in vitro, which are expected to be seed cells for the treatment of cartilage defects in cartilage tissue engineering. Appropriate growth factors are critical for the chondrocyte differentiation of synovial mesenchymal stem cells.
OBJECTIVE:To study the role of secreted factors by chondrocytes to induce chondrogenesis of synovial mesenchymal stem cells.
METHODS:The synovial mesenchymal stem cells and chondrocytes were harvested from rat knee joints and cultured through the digestion method. The supernatant was col ected from chondrocytes, and centrifuged, filtered and cryopreserved. The third passage synovial mesenchymal stem cells centrifuged as pel ets were cultured in the chondrocyte supernatant for 21 days. And the cells morphology was examined and the type II col agen and aggrecan were detected through immunohistochemistry and RT-PCR.
RESULTS AND CONCLUSION:The synovial mesenchymal stem cellpel ets cultured in the chondrocyte supernatant became cartilage-like tissue after 21 days. The type II col agen was detected positively in the matrix of synovial mesenchymal stem cellpel et immunohistochemical y. RT-PCR examination showed that the type II col agen and aggrecan expressed in the synovial mesenchymal stem cellpel et cultured in the chondrocyte supernatant. It suggested that synovial mesenchymal stem cellcould be induced to differentiate into chondrocytes depending on soluble factors secreted by chondrocytes.

BACKGROUND:Articular chondrocytes with the ability of autocrine and paracrine can provide the growth factors and microenvironment for synovial mesenchymal stem cels differentiating into the chondrocyte. The
three-dimensional scaffold could provide space for cels adhesion, proliferation and differentiation.
OBJECTIVE: To study the ability of chondrogenesis by co-culturing synovial mesenchymal stem cels and chondrocytes under the three-dimensional condition.
METHODS:The synovial membrane and articular cartilage were harvested from rat knee joint. The synovial
mesenchymal stem cels and chondrocytes were obtained through the method of enzyme digestion. The passage 3 synovial mesenchymal stem cels and passage 2 chondrocytes were co-cultured in the chitosan/I colagen
composite scaffolds at the ratio of 1:2. Then, the cels/scaffold composite was harvested to be examined
morphologicaly, histologicaly and immunohistochemicaly after being cultured 21 days. The confocal laser was also employed to detect the cels distribution in the scaffold.
RESULTS AND CONCLUSION: After being cultured 72 hours, it could be observed from the cels/scaffold composite examined through the scanning electron microscope that the cels adhered on the surface of the
scaffold and extracelular matrix surrounding the cels was seen on the scaffold. After being cultured 21 days, it could be found through the confocal laser scanning that the cels were wel-distributed on the scaffold, and cels decreased gradualy. Type II colagen was positive in the extracelular matrix immunohistochamicaly. It
suggested from this study that the synovial mesenchymal stem cels could be co-cultured with chondrocytes in the chitosan/I colagen composite scaffolds and have the ability of chondrogenesis differentiation.

BACKGROUND:Compared with other sources of mesenchymal stem cells, synovial-derived mesenchymal stem cells have significant characteristics of chondrogenesis and cloning. Therefore, synovial-derived mesenchymal stem cells are one of the most promising seed cells in cartilage tissue engineering.
OBJECTIVE:To isolate and culture synovial-derived mesenchymal stem cells of Sprague-Dawley rats, identify the multipotential differentiation and the potential ability of chondrogenic differentiation in three-dimensional culture condition.
METHODS:The synovium tissue was harvested from Sprague-Dawley rats. The synovial-derived mesenchymal stem cells were isolated with typeⅠcol agen enzyme digestion method and cultured in vitro. The passage 3 cells were detected with giemsa staining, the cellcycle, adipogenic and osteogenic differentiation were determined. The passage 3 cells were centrifuged as pel ets and cultured in the chondriogenic medium for 21 days. And the pel ets were examined by toluidine blue staining, typeⅡcol agen immunohistochemical staining and RT-PCR.
RESULTS AND CONCLUSION:The mesenchymal stem cells isolated from the synovium tissue of rats have the characteristics of mesenchymal stem cells, and exhibit fibroblast-like morphology after cultured in vitro. The multilineage differentiation potentials were also revealed. After the cellwere cultured in chondrogenic medium for 21 days, chondroid tissue was found, type II col agen and aggrecan could be detected positively by toluidine blue staining, typeⅡcol agen immunohistochemical staining, and expressed by RT-PCR examination. Therefore, synovial mesenchymal stem cells have a chondrogenic differentiation potential.

Objective To establish a method to simultaneously determinate cobalt, nickel, arsenic, molybdenum, silver, cadmium and lead in blood and urine using inductively coupled plasma tandem mass spectrometry (ICP-MS/MS). Methods The blood samples were diluted 20 times with a mixed solution of nitric acid (0.10% V/V) and Triton X-100 (0.02% V/V). The urine samples were diluted 10 times with nitric acid (1.00% V/V). Yttrium-89, rhodium-103, and lutetium-175 were used as internal standards to decrease matrix interference, and either On-Mass mode or Mass-Shift mode was used to decrease mass spectrometry interference, with detection by ICP-MS/MS. Results The linear ranges of the seven elements were 0.100-10.000 μg/L, with the correlation coefficient >0.999 9. The detection limits for the seven elements in the blood and urine were 0.003-0.021 and 0.003-0.031 μg/L, respectively, and the minimum quantification limits were 0.009-0.064 and 0.009-0.094 μg/L, respectively. The recovery rates were 96.64%-102.90% and 96.34%-104.50% for the blood and urine, respectively. The within-run relative standard deviations (RSDs) were 0.32%-2.33% and 0.25%-2.31%, and the between-run RSDs were 1.07%-3.81% and 1.30%-3.62% for blood and urine, respectively. The samples were stable at 4 ℃ for at least 14 days. Conclusion ICP-MS/MS is a simple, sensitive, and accurate method for rapid detection of seven heavy metal elements in the blood and urine of occupational hazard-exposed workers.

Objective:To explore the influence of patient participation-based dietary intervention on nutritional status for patients with severe burn.Methods:From September 2017 to January 2019, 60 severe burn patients hospitalized in the department of burn and plastic surgery of Qingdao Municipal Hospital were recruited and divided into the experimental group ( n=30) and the control group ( n=30) according to the random number table method. The control group received a regular diet. While the experimental group received a patient participation-based dietary intervention(PPDI), The wound healing time and the value of nutritional status index, such as height, body mass index(BMI), serum albumin, serum prealbumin, in the two groups on admission, at 2 weeks after intervention, 4 weeks after intervention were compared. The nutritional knowledge questionnaire of burn patients and "3-day diet diary" were used for investigation. Results:The score of nutritional knowledge showed no significant difference between the two groups before intervention. After intervention, the score of nutritional knowledge in the experimental group was 21.40±2.42, significantly higher than that in the control group (19.00±2.26) ( t value was 3.975, P<0.01). For time effect and between-group effect, there were significant difference in serum albumin between two groups ( F values were 9.232, 4.651, P<0.05); the time effect and between-group effect of serum prealbumin were statistically significant ( F values were 11.592, 6.228, P < 0.05). The wound healing time in the experimental group was significantly lower than that in the control group ( t value was -3.801, P<0.01). Conclusions:Patient participation-based dietary intervention can effectively enhanced the level of nutritional knowledge, improved nutritional status, shortened wound healing time among patients with severe burn.

Objective To assess the effect of ultrasonic thyroid artificial intelligence(AI)-assisted diagnostic system(AI-assisted diagnostic system)for diagnosing medullary thyroid carcinoma(MTC)compared with different physicians and taken papillary thyroid carcinoma(PTC)as the controls.Methods Totally 63 MTC,70 PTC and 62 benign thyroid nodules confirmed by pathology were enrolled.AI-assisted diagnostic system was utilized to analyze thyroid nodules and identify the likelihood of malignancy,and the probability value threshold was set at ≥0.40.All thyroid nodules were retrospectively reviewed and categorized by 3 physicians(1 senior physician,1 attending physician and 1 junior physician)according to Chinese thyroid imaging reporting and data system(C-TIRADS).The efficacy of AI-assisted diagnostic system and physicians for diagnosing MTC and PTC were evaluated.Results AI-assisted diagnostic system showed lower sensitivity,specificity,positive predictive value,negative predictive value,accuracy,and area under the curve(AUC)for diagnosing MTC and PTC compared with physicians.Significant differences of AUC were found between senior physician and AI-assisted diagnostic system,as well as between attending physician and AI-assisted diagnostic system for diagnosing MTC and PTC(all P＜0.01),while no significant difference of AUC was between junior physicians and AI-assisted diagnostic system(both P＞0.05).The sensitivity,specificity,positive predictive value,negative predictive value,accuracy and AUC for AI-assisted diagnostic system for diagnosing MTC were all lower than those for diagnosing PTC,but the AUC was not significantly different(P＞0.05).Conclusion Ultrasonic thyroid AI-assisted diagnostic system had relatively high value for diagnosing MTC.

Objective To construct a vimentin glycosylated mutant plasmid,and to study its impact on rat adrenal chromaffin cell (PC12) cell differentiation.Methods The vimentin glycosylated mutant plasmid was constructed,and the gel electrophoretic was performed and the sequencing identification was performed. The empty plasmid pcDNA3.1b (control group),vimentin plasmid (Vim group) and vimentin glycosylated mutant plasmid (Vim mut group) were used to transfect PC12 cells,which were cultured for 0,3 d in the con-dictions of treatment and non-treatment of exogenous oligosaccharides (Cyclo-ManN pro) respectively,and the cell differentiation records and quantitative analysis (the cell average neurite length and differentiated neu-rite cells percentage) were performed.Results A recombinant plasmid containing a glycosylation site muta-tion site 19(T-G),97(A-G),100(T-G) was successfully constructed (about 1404 bp in size).Before and after Cyclo-ManN pro treatment,the average neurite length of PC12 cells and the percentage of differentiated neu-rite cells in the Vim group were greater than those in the control group and the Vim mut group[(61.98±19.03)μm vs.(51.09±14.45)μm,(51.49±14.78)μm,(6.60±0.25)％ vs. (4.27±0.18)％,(4.76±0.33)％;(78.01±18.31)μm vs. (69.98±12.85)μm,(68.45±13.84)μm,(10.62±0.25)％ vs. (8.11±1.22)％,(5.89±0.60)％],the difference was statistically significant (P<0.05).Conclusion The glycosyla-tion modification of vimentin could be used for PC12 cell differentiation.