A variety of heat shock protein 70(HSP70)-associated therapeutic vaccines including protein vaccine,peptide vaccine,tumor cell vaccine and DNA vaccine,have been developed and applied in anti-tumor immunotherapy,in which HSP70-fused DNA vaccine attracts our attention with its superiority.It not only induces consistent anti-tumor immune response but also helps sustain immune memory.In this article we reviewed the function and application of HSP70 in the therapeutic tumor DNA vaccine.

Objective:To study the characterization of NK cell line co-transfected with hSCF and hIL-15 gene.Methods:pcDNA3-hSCF and pcDNA3-hIL-15 were transfected into NK-92 cell line with LipofectAMINETM,RT-PCR was used to identify NK-92 cell which express hSCF and IL-15,the activity of supernantes was respectively assayed by TF-1 and CTLL-2 cell line. CD3, CD16, CD56, CD25, CD48, CD54, CD69 and CD95 molecules were tested by FACS.Results:We established NK-92S15 cell line which express hSCF and hIL-15 steadily, proliferation ability demonstrated it had more extensive assembling trend,growing with enough cell number,the cytotoxicity of NK-92S15 cell was decreased compared with parental cell line when incubated with rhIL-2.CD56 and CD16 showed on difference while CD25,CD48, CD54 and CD95 decreased significantly.Conclusion:The characterization of NK-92 could be changed by co-transfecting with hSCF and hIL-15 gene, which was benefit to observe the character of NK cell activation and cytotoxicity related molecules. The gene transfection of NK-92 cell made it suitable for further study.

Objective To study the mutation of STK11 gene in a Chinese family and a sporadic patient with Peutz-Jeghers syndrome (PJS),and to provide a basis for genetic diagnosis and counseling.Methods One sporadic patient and two patients from a family with PJS were collected,all of whom had typical mucosal pigmentation and gastrointestinal polyposis.Blood samples were obtained from the two patients and six unaffected relatives in this family,the sporadic patient,and 100 healthy controls.DNA was extracted,and PCR was performed to amplify nine exons and their adjacent introns in the STK11 gene followed by direct sequencing.The sequencing results were aligned to the published sequence of STK11 gene from Genbank.Results No mutation was found in the STK11 gene of any of the patients,unaffected relatives,or healthy controls.Conclusions Genetic heterogeneity exists in Peutz-Jeghers syndrome,hinting that there may be other causative genes or sites for this entity.

Objective To discuss the clinical value of Protein induced by Vitamin K Antagonist-Ⅱ (PIVKA-Ⅱ) and alpha-Fetoproteins (AFP) in diagnosing hepatocellular carcinoma (HCC) and monitoring the treatment effects.Methods Patients were recruited by the Affiliated Hospital of Qingdao University,from August 2013 to March 2014.Serum levels of PIVKA-Ⅱ and AFP were measured by both chemiluminescence assay (CLIA) and electrochemiluminescence assay (ECLA) in patients with HCC (n =148),intrahepatic cholangiocellular carcinoma (n =37),gastric cancer and colorectal cancer (n =44),cirrhosis (n =63),chronic hepatitis B (n =38) and healthy subjects (n =57).To analyze the areas under the receiver operating characteristic curves (ROC-AUC) and to compare the sensitivity and specificity of single PIVKA-Ⅱ or AFP assay,and the combined detection.To analyze the correlation of PIVKA-Ⅱ and both tumor size and TNM staging,so do AFP,respectively.To compare the serum level changes of the two indicators in HCC patients before and after treatment.Results The serum levels of both PIVKA-Ⅱ and AFP in HCC group were higher than that in intrahepatic cholangiocellular carcinoma,gastric cancer and colorectal cancer,cirrhosis,chronic hepatitis B and healthy subjects groups (PIVKA-Ⅱ:U =866.50,424.00,958.00,292.00 and 448.00 ; AFP:U=713.00,440.50,1 182.00,614.00 and 399.00,P ＜0.001).The ROC-AUCs of the single PIVKA-Ⅱ or AFP assay and the combined detection in HCC group were not statistically different (P ＞ 0.05).The sensitivity of PIVKA-Ⅱ (87.16％) was higher than that of AFP (68.92％,x2 =4.73,P ＜ 0.05) in diagnosing HCC ; the sensitivity of the combined detection of PIVKA-Ⅱ and AFP(93.24％) was higher than that of PIVKA-Ⅱ itself (87.16％,adjusted x2 =64.70,P ＜ 0.01) ;while the specificities among them did not show statistical significance (P ＞ 0.05).Tested by Spearman rank correlation,the serum levels of PIVKA-Ⅱ and AFP were both positively related to tumor size (r =0.716,0.475 respectively,P ＜ 0.001).The serum levels of PIVKA-Ⅱ and AFP in HCC patients increased gradually correlated with tumor size (H =72.70,37.02 respectively,P ＜ 0.001) and the positive rates of PIVKA-Ⅱ and AFP were gradually improved (x2 =26.74,21.62 respectively,P ＜ 0.01),too.Based on the International TNM Staging System,the serum levels of PIVKA-Ⅱ and AFP (H =46.63,21.38 respectively,P ＜0.001) and the positive rates of PIVKA-Ⅱ and AFP (PIVKA-Ⅱ:x2 =20.40,P ＜0.01 ;AFP:x2 =8.33,P ＜0.05) in HCC patients from Ⅰ-Ⅳ stages were increased as TNM stages elevated.The serum levels of PIVKA-Ⅱ and AFP in HCC patients were both dropped sharply compared with preoperative levels (Z =-4.59,-4.22 respectively,P ＜ 0.001) and also both dropped in each of the Ⅰ-Ⅳ TNM stages (PIVKA-Ⅱ:Z =-2.85、-2.98、-2.70 respectively,P ＜ 0.05 ; AFP:Z =-2.48、-3.82、-2.50 respectively,P ＜ 0.05) compared with serum levels before treatment.Conclusion PIVKA-Ⅱ and AFP both have high clinical application values in diagnosing HCC and monitoring treatment effects.The sensitivity of PIVKA-Ⅱ in diagnosing HCC is significantly higher than AFP,and the sensitivity can be elevated by the combined detection in diagnosing HCC without reducing the specificity.

Objective To evaluate the clinical value of pro-gastrin-releasing peptide (ProGRP) for small cell lung cancer ( SCLC ).Methods Serum levels of ProGRP and neuron-specific enolase (NSE) were measured by both chemiluminescent immunoassay and electrochemiluminescent immunoassay in 46 patients with SCLC (26 patients with limited disease,20 patients with extensive disease ),51 patients with non-small cell lung cancer (NSCLC),45 patients with benign pulmonary diseases and 56 healthy subjects.Patients were recruited by the Affiliated Hospital of Medical College,Qingdao University,from September 2010 to April 2011.The receiver operating characteristic curves (ROC) was used to set the cutoff value of ProGRP and NSE and the areas under ROC ( ROC-AUC).The sensitivity and specificity of ProGRP and NSE were analyzed for diagnosing SCLC.Results Serum levels of ProGRP in healthy subjects,benign pulmonary diseases,NSCLC and SCLC groups were 22.9 ( 19.5 - 28.7 ),23.7 ( 20.0 - 27.8 ),28.9 (23.8-34.7) and 370.9( 129.4- 1951.6) ng/L respectively; the serum levels of NSE were 14.1 (12.5- 15.7),13.3(10.3- 15.3),16.8(11.7-22.1) and 39.9(16.1-93.9) μg/L,respectively.The Kruskal-Wallis H test showed significantly difference amoun groups of ProGRP and NSE (H =92.116 and 55.481,P ＜0.001 ).The serum levels of ProGRP in limited disease SCLC (LD-SCLC) group[ 156.2(65.4-547.5 ) ng/L]were also significantly higher than those in the healthy group,benign pulmonary diseases group and NSCLC group ( U =57,70 and 144,P ＜ 0.001 ).In extensive disease SCLC (ED-SCLC) group,the ProGRP and NSE results[ 1933.1 (325.9 -4512.1) ng/L and 61.0(35.4- 115.5 ) μg/L ]were higher than those in the LD-SCLC group ProGRP,NSE [ 24.3 ( 15.1 - 16.3 ) μg/L,U =119 and 153,P ＜ 0.05 ].Using healthy subjects group as control,the largest Youden index point of ROC was used to set the cut-off value of ProGRP and NSE (34.0 ng/L and 20.2 μg/L).The ROC-AUC of ProGRP (0.96 ) was statistically higher than that of NSE ( 0.86 ) in the SCLC group ( Z =2.57,P ＜0.05).The ROC-AUC results between combining detection of ProGRP and NSE (0.96 ) and ProGRP itself (0.96) were not significant difference ( Z =0.21,P ＞ 0.05 ).The sensitivity of ProGRP ( 89.1％ ) was statistically higher than that of NSE in the SCLC group (71.7％,x2 =4.90,P ＜0.05 ) ; the specificity of ProGRP (98.2％) compared with NSE did not have statistical significance (96.4％,x2 =0.00,P ＞0.05 ).The combining detection of ProGRP and NSE had no influence on the sensitivity and specificity compared with ProGRP itself (91.3％ vs 89.1％,94.6％ vs 98.2％,x2 were all 0.00,P ＞ 0.05 ).Using benign pulmonary diseases group as control,the largest Youden index point of ROC was used to set the cutoff value of ProGRP and NSE (49.5 ng/L and 23.1 μg/L).The ROC-AUC of ProGRP (0.95) was statistically higher than that of NSE (0.87) in the SCLC group (Z =1.99,P ＜0.05 ).The ROC-AUC of combining detection of ProGRP and NSE ( 0.95 ) and ProGRP itself ( 0.95 ) were not difference significantly ( Z =0.02,P ＞ 0.05 ).The sensitivity of ProGRP (84.8％ ) was statistically higher than that of NSE in the SCLC group (69.6％,x2 =4.00,P ＜0.05);the specificity of it (97.8％) was equal to that of NSE (97.8％,x2 =0.50,P ＞0.05 ).The combining detection of ProGRP and NSE had no obviously influence on the sensitivity and specificity compared with ProGRP itself ( 87.0％ vs 84.8％,95.6％ vs 97.8％,x2 were all 0.00,P ＞0.05 ).Using NSCLC group as control,the largest Youden index point of ROC was to set the cut-off value of ProGRP and NSE (49.1 ng/L and 23.0 μg/L).The ROC-AUC of ProGRP ( 0.90) was statistically higher than that of NSE (0.76) in the SCLC group (Z=2.90,P＜0.05).The ROC-AUC of combining detection of ProGRP and NSE (0.90 ) and ProGRP itself (0.90 ) were not difference significantly ( Z =0.00,P ＞ 0.05 ).The sensitivity of ProGRP ( 84.8％ ) was higher than that of NSE in the SCLC group ( 69.6％,x2 =4.00,P ＜ 0.05 ) ; the specificity of it ( 96.1％ ) was also higher than that of NSE (80.4％,x2 =6.13,P ＜ 0.05 ).The combining detection of ProGRP and NSE had no obviously influence on the sensitivity and specificity compared with ProGRP itself ( 87.0％ vs 84.8％,95.6％ vs 96.1％,x2 were all 0.00,P ＞ 0.05 ).Conclusion ProGRP has a higher diagnostic value than NSE in SCLC.