Abstract:Objective：The concept of animal organs regeneration via stem cells injection and blastocyst complementation has been proved. To apply this principle for organ regeneration，the specific organ-deficient large animals must be created firstly in order to make the specific organ ‘developmental niche’. In this study，we attempt to generate the Six1-deficient and Six1/Six4-deficient pig cell lines by CRISPR/Cas9，in order to construct pig kidney-deficient model next step. Methods：The first exon of Six1 and Six4 gene was chosen for the target to design the sgRNA and the two sgRNAs were cloned into PX330 plasmids containing Cas9 skeleton respectively. The efficiency of PX330-sgRNA vector was tested by T7EN1 enzyme assay analysis，then，the efficient PX330-sgRNA was co-transfected with the G418 resistant plasmid（pCMV-tdTomato）into the primary porcine fetal fibroblasts（PFFs）. The monoclonal cell populations were obtained through the G418 screening and the genotypes of monoclonal cells were identified by sequencing analysis. Results：Cas9/sgRNA expression vectors for Six1 and Six4 gene targeting were constructed successfully. Total 48 monoclonal cell populations with presumed Six1 gene mutation were obtained and identified by PCR，among them 21 cell colonies showed Six1 bialellic mutation（Six1-/）. By the same way，we obtained total 44 monoclonal cell populations for both Six1 and Six4 targeting and 13 of them showed Six1-/-Six4-/- genotypes. Conclusion：The Six1-/- and Six1-/- Six4-/- cell lines were obtained by highly efficient CRISPR/Cas9 targeting，which can provide the possibility in production of the pig kidney-deficient model and also be helpful for the function investigation of Six1 and Six4 genes in pig kidney development.

Abstract:Objective：To build OSBPL2-knockout porcine fetal fibroblasts（PFFs）based on CRISPR/Cas9 technology and lay the important preparative foundation for establishing an experimental animal model of deafness in miniature pigs with OSBPL2 defects. Methods：Bioinformatics methods were applied to analyze the collinearity and homology of OSBPL2 between human and pig. The secondary and tertiary structures of OSBPL2 protein of human and pig were predicted and simulated. Two single-guide RNAs（sgRNAs）respectively targeting the fifth and the sixth exon of pig OSBPL2 were designed，synthesized and cloned into pX330 plasmid. G418 was used to obtain the positive monoclonal cells after transfection into PFFs. Finally，the efficiency of CRISPR/Cas9 mediated knockout was assessed by the T7EN1 enzyme digestion assay and the genotypes of monoclonal cells were identified by sequencing analysis. Results：Bioinformatic analysis revealed that the OSBPL2 of human and pig had a good collinearity on chromosome，highly homologous amino acid sequence（88%）and similar functional domain characteristics. CRISPR/Cas9 expression vectors targeting OSBPL2 were constructed and transfected into PFFs. OSBPL2-knockout monoclonal cells with mutant genotypes were obtained by drug screening and confirmed by DNA sequencing. Conclusion：The human and pig OSBPL2 sequences and their protein structures are highly homologous. CRISPR/Cas9 expression vectors were constructed to achieve OSBPL2 gene targeting in PFFs. OSBPL2-knockout monoclonal cells were obtained，which could contribute to the construction of OSBPL2-knockout miniature pig model.

Abstract:Objective：To construct recombinant PNPLA7 shRNA adenovirus and check its knockdown efficiency in liver. Provide a new approach for further investigating the role of PNPLA7 on hepatic triglyceride metabolism. Methods： siNC and siPNPLA7 were designed and cloned into the p-shuttle-CMV-silence vector. After Sequencing identification，the plasmid was linearized by PmeⅠ and recombined with backbone pAdeasy in BJ5183 competent cells. The recombinant plasmid was linearized by PacⅠand transfected into 293A cells for packaging Ad-shPNPLA7 and Ad-shNC adenovirus. After 7 days of tail vein injection，Western blot was applied to determine the knockdown efficiency of pAdeasy-shPNPLA7 adenovirus in liver，and hepatic triglycerides content were measured. Results：The hepatic PNPLA7 protein levels were significantly decreased while the hepatic TAG levels were significantly increased in mice with tail vein injection of pAdeasy-shPNPLA7 adenovirus compared with control mice. Conclusion：The recombinant adenovirus of pAdeasy-shPNPLA7 and pAdeasy-shNC were successfully constructed. The PNPLA7 may play a role in hepatic triglycerides metabolism.

Abstract:Objective：To identify the expression of endogenous peptides during development of zebrafish and their relationship with eye development. Methods：The zebrafish embryos were collected for 0 hour and 48 hours respectively for peptide mass spectrometry analysis. The differential peptides in the development process were screened for bioinformatics analysis to detect the relationship with eye development. Results：A total of 232 bands of differential peptides were screened. Analysis of physical and chemical properties found that the molecular weight is distributed below 2 000 Da，isoelectric point of proteins mainly weak acid and weak alkaline，isoelectric point of little protein is neutral. Bioinformatics analysis found that Vitellogenin（VTG），Titin（TTN） encoded precursor protein have large numbers of peptides. The molecular function and physiological function of all differential peptides were analyzed by ingenuity pathway analysis（IPA），which found they are closely related embryonic development. The signal pathway analysis revealed that axonal-mediated signaling pathway，FAK signal pathway and MAPK signal pathway related to eye development. Conclusion：The differential endogenous peptides of zebrafish embryo development are stable，closely related to embryonic development，and may affect eye development through related signal pathway，which will provide new research ideas and mechanism for eye development.

Abstract:Objective：To construct the recombinant plasmid containing SRY related high mobility group box 18（SOX18）gene and investigate the effect of the recombinant protein on angiogenesis of vascular endothelial cells. Methods：The whole sequence of SOX18 gene that amplified from human umbilical vein endothelial cells（HUVEC）by reverse transcription-polymerase chain reaction（RT-PCR）were inserted into the expression plasmid pSecTag-2B to construct the recombinant vector，and termed as pST-SOX18. pST-SOX18 was then transfected into the HEK293T and vascular endothelial cells，respectively. Subsequently，Western blotting was carried out to determine the expression of the recombinant protein. CCK-8 assay was used to assess the effect of pST-SOX18 on proliferation of vascular endothelial cells. Results：The recombinant plasmid pST-SOX18 was constructed successfully. pST-SOX18 was successfully transfected into HEK293T and vascular endothelial cells，target protein was determined by immunoblotting. pST-SOX18 transfection significantly promoted vascular endothelial cell proliferation in vitro. Conclusion：The recombinant plasmid expressing functional SOX18 was constructed successfully. SOX18 have a potent potential in promoting the proliferation of vascular endothelial cells.

Abstract:Objective：The purpose of this study was to investigate the role of autophagy in oxygen-glucose-deprivation/reoxygenation（OGD/R）injury in rat neurons. Methods：Cortical neurons were isolated from Sprague-Dawley rats and identified by immunofluorescence. The cortical neurons were randomly assigned to four groups：control group（Ⅰ），experimental group（OGD/R group，Ⅱ），JNK inhibitor pretreatment group（Ⅲ）and JNK inhibitor pretreatment+OGD/R group（Ⅳ）. Results：Neuronal cell viability significantly decreased after 6　h and 12　h of reoxygenation in Group Ⅳ（P < 0.05）. Electron microscopy results showed the presence of many autophagic vacuoles and the formation of autolysosomes in the neurons；the number of autophagic vacuoles decreased transiently at 6　h，while a new autophagic flux and a large number of empty autophagic vacuoles were observed at 12　h. In Group Ⅳ，a large number of autophagic vacuoles were present at 0.5　h and 2　h of reoxygenation，which gradually decreased with increasing reoxygenation time. No significant differences in the expression of the LC3Ⅱprotein were detected between the GroupⅡ and Ⅳ prior to 6　h of reoxygenation，and LC3Ⅱ expression showed an overall rise-decline pattern. However，LC3Ⅱ protein expression increased in Group Ⅱ at 12　h of reoxygenation，whereas a continuous decline was observed in Group Ⅳ. The levels of phosphorylated JNK and Bcl-2 and the expression of Beclin-1 increased gradually as the reoxygenation time going in Group Ⅱ，whereas they increased at 12　h of reoxygenation in Group Ⅳ（P < 0.05）. In addition，progressive dissociation of the Bcl-2/Beclin-1 complex was observed in the Group Ⅱ，while JNK inhibitor suppressed this dissociation. Conclusion：The regulation of the JNK/Bcl-2/Beclin-1 signaling pathway may be one of the mechanisms underlying the OGD/R-induced autophagic cell death of neurons.

Abstract:Objective：The aim of our research is to investigate the role of oxidative stress in cellular aging of human epidermal melanocytes. Methods：Cultured human melanocytes were subjected to different doses of H2O2（0、200、400、600、800、1 000 μmol/L）for 5 d，after which cell viability and apoptosis were assessed.Next we investigated whether exposure to low levels of H2O2（0~400 μmol/L）induces premature senescence via evaluating senescence-associated β-galactosidase staining，cell proliferation assay，cell cycle analysis，E-cadherin and senescent gene expression，etc. Results：Cell viability and apoptosis in melanocytes cultured with H2O2 at 200~400 μmol/L showed no significant difference as compared with cells cultured without H2O2 and apoptosis of melanocytes progressively increased at high concentrations of H2O2 （600~1 000 μmol/L）. Treatment with 400 uM H2O2 tended to exhibit dendrite retraction，increased SA-beta-gal activity，declined rate of proliferation，G2 cell cycle arrest，and changes in the expression levels of p53 and p21.We further show that pre-senescent melanocytes express markedly reduced levels of E-cadherin. Conclusion：Melanocytes undergo premature senescence under the stimulation of 400 μmol/L concentration of H2O2 .

Abstract:Objective：To investigate protectory effects and possible mechanisms of lipoxin A4（LXA4）on the type Ⅱ alveolar epithelial cell line MLE-12 with hyperoxia injury. Methods：MLE-12 cells were cultured and divided into five groups randomly：I：air group，Ⅱ：hyperoxia group，Ⅲ：hyperoxia+ LXA4 group，Ⅳ：hyperoxia+ TGF-β1 neutralizing antibodies，Ⅴ：hyperoxia+ TGF-β1 neutralizing antibodies+ LXA4. The growth state and morphological changes of the cells were observed by the inverted phase microscope；the mRNA expression of collagen Ⅰ，tenascin-C，TGF-β1 receptor（TGF-βR1），TGF-βR2 and Smad3 were measured by real-time quantitative PCR；the protein levels of TGF-β1/Smads signaling pathway（TGFβR1，Smad2，Smad3，Smad4，p-Smad2 and p-Smad3）were detected by Western blot. Results：①Morphological change：the cells under hyperoxia lost their normal shapes and showed necrosis，while the cells cultured with LXA4 and/lor TGF-β1 neutralizing antibodies were normal appropriately. ②The mRNA expression of extracellular matrix（ECM）：LXA4 and TGF-β1 neutralizing antibodies attenuated the mRNA expression of collagenⅠ，tenascin-C markedly（P < 0.05），which expressed in the group hyperoxia+TGF-β1 neutralizing antibodies + LXA4 decreased most obviously. ③The mRNA and protein levels of TGF-β1/Smads signaling pathway：Compared with that of the hyperoxia group，the mRNA expression of TGF-βR1，TGF-βR2 and Smad3 decreased（P < 0.05），while the protein level of TGF-β1/Smads signaling pathway had the same change in intervention group，and it in the the group hyperoxia+ TGF-β1 neutralizing antibodies +LXA4 decreased most obviously（P < 0.05）. Conclusion：LXA4 can achieve protection of hy-peroxia-induced injury to MLE-12 which may be related to collagenⅠ， tenascin-C and TGF-β1 /Smads signaling pathway.

Abstract:Objective：To validate PDCD4 expression in male mouse germ cells. Methods：We verified Pdcd4 expression in testes of different age mice via RT-PCR and Western blot. The localization of PDCD4 in male reproductive ridge and testis in different age groups was detected by immunofluorescence. Results：RT-PCR and Westen bolt results showed that PDCD4 was expressed in the 0-week-old mouse testes and continued to express till adulthood； Immunofluorescence showed that PDCD4 was expressed in embryonic male germ cells. PDCD4 was specifically expressed in spermatogonia in the testis of 0 week and 1 week old mice after birth；In the 2-week-old mouse testes，PDCD4 was distributed in spermatogonia and spermatocytes；In the 3 weeks，4 weeks and 5 weeks old mice testicular tissue，PDCD4 mainly distributed in spermatogonia，spermatocytes and round spermatids. Conclusion：PDCD4 is distributed in each level of male germ cells，which lay the foundation for further study of the role of PDCD4 in spermatogenesis.

Abstract:Objective：To explore the association between single nucleotide polymorphisms（SNPs）in the Wnt signaling pathway genes and the curative effect and prognostic of metastatic colorectal cancer（mCRC）patients who received irinotecan-based chemotherapy. Methods：We selected seven key genes in the Wnt signaling pathway and genotyped the SNPs within seven genes among 110 mCRC patients treated with irinotecan-based chemotherapy. Logistic regression and cox regression analysis were performed to evaluate the association between SNPs and the curative effect and prognostic of mCRC patients. Results：Patients with the LRP6 rs10772542 C alleles had a lower disease control rate（DCR）compared with those with T alleles（OR=2.49，95%CI=1.28~4.83，P=0.007）. LEF1 rs749414 G alleles had a longer progression-free survival（PFS）compared with the T alleles（HR=0.55，95%CI=0.40~0.78，P=0.001）and WNT7B rs10448605 T alleles had a shorter PFS compared with the C alleles（HR=1.65，95%CI=1.13~2.41，P=0.009）. Furthermore，WNT2 rs2239957 C alleles had a longer overall survival（OS）compared with the G alleles（HR=0.54，95%CI=0.35~0.85，P=0.007）. Conclusion：Genetic variants in the Wnt signaling pathway genes contribute to the curative effect and prognostic of mCRC patients who received irinotecan-based chemotherapy.

Abstract:Objective：To investigate and establish the microscopic features of colorectal diseases via multiphoton microscopy（multiphoton microscopic imaging，MPM）initially，evaluating the diagnostic value in vitro. Methods：The multiphoton images of 100 fresh tissues confirmed pathologically colorectal normal tissues，hyperplastic polyps，adenomas and adenocarcinomas were collected and retrospectively analyzed to establish diagnostic standards with MPM. Results：Through the investigation，we found the colorectal normal，hyperplastic polyps，adenoma and adenocarcinoma were different in the crypt opening，glandular structure，epithelial cells and collagen fibers. Besides，the cellular areas by quantitive analysis were 32.14 ± 1.96 μm2、32.69 ± 2.34 μm2、55.40 ± 4.63 μm2、71.83 ± 5.30 μm2、85.90 ± 11.50 μm2，respectively. P values were both of statistically significance. Conclusion：The real-time multiphoton microscopic imaging can be used to classify and grade the colorectal diseases safely and effectively with high resolution. It is potential to become the new generation of optical technique via integrating with the endoscopes in the future.

Abstract:Objective：To investigate the clinical features and possible mechanisms of attention function in patients with episodic headache and chronic headache. Methods：Through a total of 18 patients with Episodic Headache（EH），20 patients with chronic headache（CH）and 21 healthy controls，respectively，neuropsychological assessment including mini mental state assessment scale（mini mental state exam，MMSE），Montreal cognitive assessment（China - Beijing edition Beijing Chinese version of Montreal Cognitive Assessment，MoCA- C），digital span test（digit span test，DS），frontal lobe function rating scale（frontal assessment battery，FAB），the hospital anxiety and Depression Scale（hospital Anxiety and depression subscales，HADS）and Pittsburgh Sleep Scale（Pittsburgh sleep quality index，PSQI）and attention network test（attention networks test，ANT），and comparative analysis. Results：There were significant differences in MoCA- C（P< 0.001 or P< 0.001），DS（P=0.002 or P=0.004），HADS（anxiety：P< 0.001 or P=0.002；depression：P< 0.001 or P=0.001）and PSQI（P< 0.001 or P=0.001）between chronic headache and episodic headache or healthy controls. Significant difference was also found in FAB between chronic headache and healthy control（P=0.021）. ANT showed that there were no significant differences in alerting，orienting or accuracy among three groups，but median response time was prolonged significantly in patients with chronic headache than those with episodic headache or healthy controls（P=0.002 or P=0.035），and compared with healthy controls，executive control function was also disrupt in chronic headache patients（P=0.042）. Conclusion：Headache does disrupt attention function，especially in chronic headache，mainly for the average reaction time to perform tasks related to CH patients with prolonged attention，executive function decreased，suggesting that CH damaged the attention network-executive function，and these might be one of the key mechanisms of headache disrupting attention.

Abstract:Objective：To investigate multiple apparent diffusion coefficient（ADC）histogram parameters that show correlations with different subtypes of invasive ductal carcinoma. Materials and Methods：From March 2014 to March 2016，a total of 182 invasive ductal carcinomas were included in this retrospective study. Various ADC histogram parameters were calculated including the entire tumor volume：the ADCmin，ADC10，ADCmean，ADCmedian，ADC90，ADCmax，skewness and kurtosis. The ADC multiple parameters of the histogram were calculated through Firevoxel software. The normality and homoscedasticity of imaging measures were tested using the Levene’s tests. Data satisfying the assumption were subjected to ANOVA and independent-sample t test. Conversely，data not satisfying the assumption were analyzed by using the Kruskal-Wallis test and Mann-Whitney U test. The correlation between ADC parameters with different subtypes of invasive ductal carcinoma were analyzed. Results：For most ADC parameters except for ADCmin，the mean of variable ADC parameters of HER-2 positivity，Luminal A，triple-negative，and Luminal B diseases were arranged in descending order（e.g. 1.501×10-3，1.401×10-3，1.330×10-3，1.223×10-3 mm2/s in ADC90，respectively）with statistical significant difference（P=0.019，0.035，< 0.001，respectively）. Conclusion：Various ADC parameters were correlated with prognostic factors and subtype，except for ADCmin. HER-2 positivity showed high ADC values than other subgroups.

Abstract:Objective：To evaluate the diagnostic value of 64-row spiral CT in the preoperative TN staging of gastric cancer and the influence of water-filling method on the accuracy of the preoperative TN staging. Methods：The retrospective analysis was conducted to compare the CT diagnosis and pathological data of 120 gastric cancer patients（water-drinking group：60 cases；no water-drinking group：60 cases）. Results：In the water-drinking group，there was high consistency between CT diagnosis of T staging and pathological data（Kappa=0.714）. In the no water-drinking group，there was medium consistency between CT diagnosis of T staging and pathological data（Kappa=0.506）. The overall accuracies in two groups had significant difference（P=0.039）. In the water-drinking group，there was high consistency between CT diagnosis of N staging and pathological data（Kappa=0.640）. In the no water-drinking group，there was medium consistency between CT diagnosis of N staging and pathological data（Kappa=0.510）. The overall accuracies in two groups had no significant difference（P=0.426）. Conclusion：There is a higher clinical value of application with 64-row spiral CT in the preoperative staging of gastric cancer，and drinking water before the CT scanning could increase the accuracy of the preoperative T staging，which has certain guiding significance on the choice of therapeutic regimen for gastric cancer.

Abstract:Objective：To analyze the level of circulating endothelial progenitor cells（cEPCs）in patients with stable chronic obstructive pulmonary disease（COPD）. Methods：Major online database including Pubmed，Embase，Web of science were searched to find published case-control studies on the association between cEPCs and patients with stable COPD. The results were analyzed using Stata14 software. Results：A total of 12 studies containing 550 subjects were enrolled. Meta analysis showed that the level of cEPCs was significantly declined in patients with stable COPD（standardized mean difference（SMD）= -1.38，95% confidence interval（CI）（-2.01，-0.74），Z=4.23（P < 0.001）. Subgroup analysis indicated that using flow cytometry the level of cEPCs was remarkable decresed in patients with stable COPD than normal controls；however，there is no statistic significance in the level of cEPCs between patients with stable COPD and controls by counting EPCs colony forming units. By sensitivity analysis，funnel plot，and “trim and fill”，it showed that the results were robust. Conclusion：The level of cEPCs is low in patients with stable COPD compared with healthy controls，especially by the method of flow cytometry，which may lead to the deficient capacity of endothelial repairing.

Abstract:Objective：This systematic review and Meta-analysis aimed to investigate the metastatic rate of hilar lymph node and mediastinal lymph node in clinical stage Ⅰ non-small-cell lung cancer patients with either pure ground-glass opacity（Pure-GGO）or mixed ground-glass opacity（Mixed-GGO），therefore to indicate how to dissect lymph nodes in patients with GGO. Methods：A systematic search of the published literature was conducted using the main databases（Science Direct，PubMed，Springer Link and Wiley Online Library）to collect relevant case-control studies that compared Pure-GGOs and Mixed-GGOs in clinical stage I non-small-cell lung cancer patients. Meta-analysis was performed extracting data from the published literature using STATA 12.0. The results of the Meta-analysis were expressed as an odds ratio（OR）and their corresponding 95% confidence interval（CI）. Results：We extracted data from three case-control studies，with a total of 736 patients. There was no significant difference（OR=3.66，95% CI：0.68-19.58，P=0.13）in the rates of metastases in all lymph nodes in patients with either Pure-GGO or Mixed-GGO. In addition，there were no significant differences（OR=4.22，95% CI：0.77-23.19，P=0.10）in the rates of metastases in hilar lymph nodes in patients with either Pure-GGO or Mixed-GGO，but one study showed an OR of 20.18（95% CI：0.94-432.12）. There was no significant difference in rates of metastases of mediastinal lymph nodes in patients with either Pure-GGO or Mixed-GGO（OR=1.10；95% CI：0.19-6.32，P=0.92）. Conclusion：The results indicated no statistically significant difference in metastatic rates of hilar lymph nodes and mediastinal lymph nodes in patients with either Pure-GGO or Mixed-GGO. However，as thoracic surgeon，we must be particularly cautious about metastasis in hilar lymph nodes in patients with Mixed-GGO.