Objective To explore the application value of plasma sHLA-G in diagnosis of CIN and cervical cancer. Methods The plasma sHLA-G levels were detected by ELISA in 102 cases with cervical cancer( FIGO Ⅰ stage 32 cases, Ⅱ stage 28 cases, Ⅲ stage 25 cases and Ⅳstage 17 cases; tumor size:＜4 cm 63 cases and ≥4 cm 39 cases; squamous cell carcinoma 78 cases and adenocarcinoma 24 cases;cell differentiation:well 57 cases, moderate 29 cases and poor 16 cases; lymph nodes metastasis negative64 cases and positive 38 cases ), 72 cases with CIN( Ⅰ grade 21 cases, Ⅱ grade 25 cases and Ⅲ grade26 cases ) and 20 cases of healthy controls. The diagnostic value of sHLA-G and its correlations with clinical parameters were analyzed. Results The plasma levels of sHLA-G were 193.6( 151.3-287.4 ) kU/L in cervical cancer group, 48.3( 34.6-57.2 ) kU/L in CIN Ⅰ group, 91.3( 68.2-118.6 ) kU/L in CIN Ⅱ group, 106.4( 73.8-165.7 ) kU/L in CIN Ⅲ group and 45.2( 38.0-55.5 ) kU/L in health control group.The level of sHLA-G was significantly higher in cervical cancer group than that in CIN Ⅰ group, CIN Ⅱ group, CIN Ⅲ group and healthy control group( U value of 8.832, 6.456, 4.017, 9.873, P ＜ 0.05,respectively ). The level of sHLA-G was significantly higher in CIN Ⅱ group and CIN Ⅲ group than that in CIN Ⅰ group and health control group( U value of 4.361,4.892, 5.139, 5.485, P ＜0.05, respectively ).The levels of SCC Ag in healthy control group, CIN Ⅰ group, CIN Ⅱ group, CIN Ⅲ group and cervical cancer group were 0.43( 0.38-0.69 )μg/L, 0.47( 0.35-0.72 )μg/L, 0.65( 0.53-0.81 )μg/L, 0.82( 0.54-1.03 )μg/L and 1.02( 0.62-1.87 )μg/L. The level of SCC-Ag was significantly higher in cervical cancer group than that in CIN Ⅰ group, CIN Ⅱ group and healthy control group( U value of 7.926, 4.877, 8.132,P ＜0.05, respectively ). The level of SCC-Ag was significantly higher in CIN Ⅲ group than that in CIN Ⅰ group and health control group( U value of 6.574, 6.763, P ＜0.05, respectively ). The levels of CA125 in healthy control group, CIN Ⅰ group, CIN Ⅱ group, CIN Ⅲ group and cervical cancer group were 14.38 ( 6.14-21.82 ) kU/L, 15.42( 6.25-23.53 ) kU/L, 21.34( 9.82-32.58 ) kU/L, 25.69( 14.47-38.71 )kU/L and 27.72( 14.29-43.87 ) kU/L. The level of CA125 was significantly higher in cervical cancer group than that in CIN Ⅰ group, CIN Ⅱ group and healthy control group( U value of 7.564, 4.522, 7.429, P ＜0.05, respectively ). The level of CA125 was significantly higher in CIN Ⅲ group than that in CIN Ⅰ group and health control group( U value of 5.871, 5.435, P ＜0.05, respectively ). ROC curve analysis showed AUC for sHLA-G was 0.828( 95% CI:0.768-0.879 ), which was high as compared with the AUC of SCC-Ag [ 0.727( 95% CI:0.658-0.788 );Z = 2.294, P ＜ 0.05 ] and the AUC of CA125 [ 0.705( 95% CI:0.636-0.769 );Z =2.842 ,P ＜0.05 ]. There was no significant difference of diagnostic efficiency between SCC and CA125( Z =0.672, P ＞ 0.05 ). When cutoff value of sHLA-G was 109.6 kU/L, the diagnostic sensitivity,specificity, positive predictive value, negative predictive value and accuracy rate were 86.3%, 76.1%,80.0%, 83.3%, and 78.4%, respectively. The levels of sHLA-G in cervical cancer patients were significantly correlated with FIGO stages and lymphoid node metastasis ( U value of 6.085, 4.451, P ＜0.05, respectively ), while there were no significant differences between the levels of sHLA-G and age,tumor size, histological type and cell differentiation( U value of 1.274, 1.956, 1.268, 2.719, P ＞0.05,respectively ). Conclusions sHLA-G can be used for the early screening of cervical cancer and its precancerous lesion. It could also be used as an index for judging progression and lymphoid node metastasis.

As main recipient cells for porcine reproductive and respiratory syndrome virus (PRRSV), porcine alveolar macrophage (PAM) are involved in the progress of several highly pathogenic virus infections. However, due to the fact that the PAM cells can only be obtained from primary tissues, research on PAM-based virus-host interactions remains challenging. The improvement of induced pluripotent stem cells (iPSCs) technology provides a new strategy to develop IPSCs-derived PAM cells. Since the CD163 is a macrophage-specific marker and a validated receptor essential for PRRSV infection, generation of stable porcine induced pluripotent stem cells lines containing CD163 reporter system play important roles in the investigation of IPSCs-PAM transition and PAM-based virus-host interaction. Based on the CRISPR/Cas9- mediated gene editing system, we designed a sgRNA targeting CD163 locus and constructed the corresponding donor vectors. To test whether this reporter system has the expected function, the reporter system was introduced into primary PAM cells to detect the expression of RFP. To validate the low effect on stem cell pluripotency, we generated porcine iPSC lines containing CD163 reporter and assessed the pluripotency through multiple assays such as alkaline phosphatase staining, immunofluorescent staining, and EdU staining. The red-fluorescent protein (RFP) expression was detected in CD163-edited PAM cells, suggesting that our reporter system indeed has the ability to reflect the expression of gene CD163. Compared with wild-type (WT) iPSCs, the CD163 reporter-iPSCs display similar pluripotency-associated transcription factors expression. Besides, cells with the reporter system showed consistent cell morphology and proliferation ability as compared to WT iPSCs, indicating that the edited-cells have no effect on stem cell pluripotency. In conclusion, we generated porcine iPSCs that contain a CD163 reporter system. Our results demonstrated that this reporter system was functional and safe. This study provides a platform to investigate the iPS-PAM development and virus-host interaction in PAM cells.
Swine ; Animals ; Induced Pluripotent Stem Cells/metabolism* ; Receptors, Cell Surface/genetics* ; Antigens, CD/metabolism* ; Porcine respiratory and reproductive syndrome virus/genetics*