ObjectiveTo investigate the effect and mechanism of transplantation of human umbilical cord mesenchymal stem cell （hUC-MSC） with overexpression of the Numb gene in the treatment of cholestatic liver fibrosis （CLF）. MethodsThe technique of lentiviral transfection was used to induce the overexpression of the Numb gene in hUC-MSC （hUC-MSC^Numb-OE）， and hUC-MSC transfected with empty vector （hUC-MSC^OE-EV） was used as negative control. Bile duct ligation （BDL） was performed to establish a rat model of CLF， and then the rats were randomly divided into BDL group， hUC-MSC group， hUC-MSC^OE-EV group， and hUC-MSC^Numb-OE group， while a sham-operation group was also established. The rats in the intervention groups were given a single splenic injection of the corresponding cells after BDL， and samples were collected at the end of week 4. Related indicators were measured， including serum biochemistry， liver histopathology， the content of hydroxyproline （Hyp） in the liver， hepatic stellate cell activation， ductular reaction， liver regeneration， and the expression levels of key molecules in the Numb-p53 signaling axis. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the least significant difference t-test was used for further comparison between two groups. ResultsCompared with the BDL group， the hUC-MSC group and the hUC-MSC^OE-EV group had significant reductions in the levels of serum biochemical parameters （aspartate aminotransferase， gamma-glutamyl transpeptidase， total bile acid， total bilirubin， and direct bilirubin）， liver fibrosis markers （the content of Hyp and the expression levels of alpha-smooth muscle actin， tumor necrosis factor-α， and transforming growth factor-beta 1）， and ductular reaction markers （the expression levels of CK7 and CK19） （all P <0.05）， and compared with the hUC-MSC^OE-EV group， the hUC-MSC^Numb-OE group had significantly greater improvements in the above indicators （all P <0.05）. In addition， compared with the hUC-MSC^OE-EV group， the hUC-MSC^Numb-OE group had significant improvements in the expression levels of liver regeneration-related markers （albumin and hepatocyte nuclear factor 4α） and the molecules associated with the Numb-p53 signaling axis （Numb， pNumb， Mdm2， and p53） （all P <0.05）. ConclusionOverexpression of the Numb gene can enhance the therapeutic effect of hUC-MSC on CLF， possibly by activating the Numb-PTB^L-p53-HNF4α axis， promoting the hepatic differentiation of hUC-MSCs and subsequently enhancing liver regeneration.

ObjectiveTo investigate the effect of transplantation of bone marrow mesenchymal stem cells （BMSCs） co-cultured with bone marrow-derived M2 macrophages （M2-BMDMs）， named as BMSC^M2， on a rat model of liver cirrhosis induced by carbon tetrachloride （CCl₄）/2-acetaminofluorene （2-AAF）. MethodsRat BMDMs were isolated and polarized into M2 phenotype， and rat BMSCs were isolated and co-cultured with M2-BMDMs at the third generation to obtain BMSC^M2. The rats were given subcutaneous injection of CCl₄ for 6 weeks to establish a model of liver cirrhosis， and then they were randomly divided into model group （M group）， BMSC group， and BMSC^M2 group， with 6 rats in each group. A normal group （N group） with 6 rats was also established. Since week 7， the model rats were given 2-AAF by gavage in addition to the subcutaneous injection of CCl₄. Samples were collected at the end of week 10 to observe liver function， liver histopathology， and hydroxyproline （Hyp） content in liver tissue， as well as changes in the markers for hepatic stellate cells， hepatic progenitor cells， cholangiocytes， and hepatocytes. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the least significant difference t-test was used for further comparison between two groups. ResultsCompared with the N group， the M group had significant increases in the activities of serum alanine aminotransferase （ALT） and aspartate aminotransferase （AST） （P<0.01）； compared with the M group， the BMSC and BMSC^M2 groups had significant reductions in ALT and AST （P<0.01）， and the BMSC^M2 group had significantly better activities than the BMSC group （P<0.05）. Compared with the N group， the M group had significant increases in Hyp content and the mRNA and protein expression levels of alpha-smooth muscle actin （α-SMA） in the liver （P<0.01）； compared with the M group， the BMSC and BMSC^M2 groups had significant reductions in Hyp content and the expression of α-SMA （P<0.05）， and the BMSC^M2 group had a significantly lower level of α-SMA than the BMSC group （P<0.01）. Compared with the N group， the M group had significant increases in the mRNA expression levels of the hepatic progenitor cell markers EpCam and Sox9 and the cholangiocyte markers CK7 and CK19 （P<0.01） and significant reductions in the expression levels of the hepatocyte markers HNF-4α and Alb （P<0.01）； compared with the M group， the BMSC and BMSC^M2 groups had significant reductions in the mRNA expression levels of EpCam， Sox9， CK7， and CK19 （P<0.05） and significant increases in the mRNA expression levels of HNF-4α and Alb （P<0.05）， and compared with the BMSC group， the BMSC^M2 group had significant reductions in the mRNA expression levels of EpCam and CK19 （P<0.05） and significant increase in the expression level of HNF-4α （P<0.05）. ConclusionM2-BMDMs can enhance the therapeutic effect of BMSCs on CCl₄/2-AAF-induced liver cirrhosis in rats， which provides new ideas for further improving the therapeutic effect of BMSCs on liver cirrhosis.

Objective To investigate the effect and mechanism of Yiguan Decoction(YGJD)on the efficacy of M1 bone marrow-derived macrophages(M1-BMDMs)in the treatment of rats with liver cirrhosis induced by 2-AAF/CCl4.Methods BMDMs were isolated and induced into M1-BMDMs by lipopolysaccharide.A total of 50 male Wistar rats were randomly divided into normal group with 5 rats and model group with 45 rats.The rats for modeling were given subcutaneous injection of 50%CCl4 twice a week.Since week 7,the rats for modeling were randomly divided into model group(M group),YGJD group,M1-BMDM group,M1-BMDM+YGJD group,and sorafenib(SORA)group,and they were given subcutaneous injection of 30%CCl4 to maintain the progression of liver cirrhosis and intragastric administration of 2-AAF.CCR2 inhibitors were added to the drinking water,and each group was given the corresponding intervention.Related samples were collected at week 9.The rats were observed in terms of serum liver function parameters,liver pathology,hydroxyproline(Hyp)content in liver tissue,hepatic stellate cell activation,hepatic fibrosis and inflammation factors,and the expression levels of molecules associated with the Wnt signaling pathway.A one-way analysis of variance was used for comparison of continuous data between multiple groups,and the least significant difference t-test was used for further comparison between two groups.Results Compared with the M group,the M1-BMDM+YGJD group had significant reductions in the serum levels of alanine aminotransferase,aspartate aminotransferase,and total bilirubin(TBil)(all P<0.05)and a significant increase in the content of albumin(Alb)(P<0.05),and compared with the M1-BMDM group,the M1-BMDM+YGJD group had a significant reduction in the serum level of TBil(P<0.05)and a significant increase in the serum level of Alb(P<0.05).Compared with the M1-BMDM group,the M1-BMDM+YGJD group had significant reductions in the expression levels of CD68 and TNF-α(P<0.05).Compared with the M1-BMDM group,the M1-BMDM+YGJD group had significant reductions in Hyp content and Sirius red positive area(P<0.05).As for the non-canonical Wnt signaling pathway molecules,compared with the M1-BMDM group,the M1-BMDM+YGJD group had significantly lower mRNA and protein expression levels of Wnt5a(P<0.05)and mRNA expression level of Fzd2(P<0.05),as well as significant reductions in the mRNA expression levels of Wnt4,Wnt5b,and Fzd3(P<0.05),while there were no significant changes in the mRNA expression levels of the canonical Wnt signaling pathway molecules β-catenin,LRP5,LRP6,Fzd5,and TCF.Conclusion YGJD can enhance the therapeutic effect of M1-BMDMs on rats with liver cirrhosis induced by 2-AAF/CCl4,possibly by inhibiting the non-canonical Wnt5a/Fzd2 signaling pathway,which provides new ideas for the synergistic effect of traditional Chinese medicine on M1-BMDMs in the treatment of liver cirrhosis.

Objective:To investigate the interventional effect of bone marrow mesenchymal stem cell (BMMSC) transplantation with different doses of X-ray irradiation induced hepatic injury in mice.Methods:Eighteen female C57BL/6J mice were randomly divided into 0, 2, and 3 Gy irradiation groups and 0, 2, and 3 Gy transplantation groups. The irradiation group was used as the control and injected with an equal volume of culture medium. The mice in the transplantation group were irradiated with different doses of X-ray irradiation, and BMMSCs were intravenously infused into the bone marrow. The mice were sacrificed for sampling at the end of the 21st day. Mice body weight changes were recorded daily. The changes in the content of peripheral blood lymphocytes, red blood cells, platelets, and hemoglobin were detected by an automatic blood tester. The morphological changes in mice liver tissues were observed by hematoxylin-eosin staining. The serum activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected by a biochemical analyzer. The reduced glutathione contents in liver tissue were detected by the microplate method. The malondialdehyde content in liver tissue was detected by thiobarbituric acid. The content of total superoxide dismutase (T-SOD) in liver tissue was detected by the hydroxylamine method. The expression of the F4/80 protein in liver tissue was detected by the immunohistochemistry method. The protein expression of nuclear transcription factor erythroid 2 related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) in liver tissue was determined by the western blotting method. The mRNA expression of NLRP3, IL-6, and Nrf2 in liver tissue was detected by a real-time quantitative polymerase chain reaction. The multiple-group comparisons were analyzed by factorial analysis of variance. The inter-group comparisons were analyzed by the LSD method for statistical analysis.Results:The contents of peripheral blood lymphocytes, erythrocytes, platelets, and hemoglobin were significantly decreased in the 3 Gy irradiation group than the 0 Gy irradiation group ( P<0.05), while the activities of serum ALT and AST were significantly increased ( P<0.05). The malondialdehyde content, F4/80 protein expression level, nucleotide-binding domain and leucine-rich repeats, nucleotide oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3), and interleukin 6 mRNA expression levels were significantly increased in liver tissue, while the contents of T-SOD and glutathione, Nrf2 and HO-1 protein expression levels, and Nrf2 mRNA expression level in liver tissue were significantly decreased ( P<0.05). The contents of peripheral blood lymphocytes, red blood cells, platelets, and hemoglobin were significantly increased in the 3 Gy transplantation group than the 3 Gy irradiation group ( P<0.05), while the activities of serum ALT and AST were significantly decreased ( P<0.05). The malondialdehyde content, F4/80 protein expression level, NLRP3 and interleukin-6 mRNA expression levels in liver tissue were significantly decreased ( P<0.05), while the content of T-SOD and glutathione, Nrf2 and HO-1 protein expression levels, and Nrf2 mRNA expression level in liver tissue were significantly increased ( P<0.05). Conclusion:X-ray irradiation at a dose of 3 Gy can induce liver oxidative damage in mice. BMMSC transplantation can improve X-ray irradiation-induced liver oxidative damage in mice, and its mechanism of action may be related to the regulation of the Nrf2/HO-1 pathway.

Objective:To investigate whether the overexpression of Numb gene can effectively intervene the progression of cholestatic liver fibrosis (CLF) in adult liver.Methods:Twenty-four SD rats were randomly divided into sham operation (Sham, n=6), common bile duct ligation (BDL, n=6), empty vector plasmid (Numb-EV, n=6) and numb gene overexpression group (Numb-OE, n=6). The CLF model was prepared by common bile duct ligation. Simultaneously, the model was established, and the adeno-associated virus (AAV) carrying the cloned numb gene was injected into the rats' spleens. Samples were collected at the end of four weeks. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (Alb), serum total bilirubin (TBil), serum total bile acid (TBA), liver histopathology, liver tissue hydroxyproline (Hyp) content, and alpha smooth muscle actin (α-SMA), cytokeratin (CK) 7, and CK19 expression conditions were determined in liver tissue. An analysis of variance was used to compare the means of multiple groups. Results:Compared with the Sham group, the Numb mRNA level in the rat liver tissue was significantly decreased in the BDL group (0.872±0.237 vs. 0.452±0.147, P=0.003). Compared with the Numb-EV group, the Numb mRNA level in the liver tissue was significantly increased in the Numb-OE group (0.487±0.122 vs. 1.094±0.345, P＜0.01). Compared with the Sham group, the Hyp content (μg/L) (288.46±49.49 vs. 901.98±271.85, P＜0.01) and the α-SMA mRNA level (0.858±0.234 vs. 8.976±1.398, P＜0.01) were significantly higher in the BDL group. Compared with the Numb-EV group, the Hyp content (864.32±113.54 vs. 580.44±171.77, P=0.039), the α-SMA mRNA level (6.138±1.443 vs. 1.322±0.859, P＜0.01) and the protein levels were significantly reduced in the Numb-OE group. Compared with the Sham group, the serum ALT, AST, TBil, and TBA levels were significantly increased in the BDL group ( P＜0.01), and the ALB content was significantly decreased ( P＜0.01). Compared with the Numb-EV group, AST and TBil levels were significantly reduced in the Numb-OE group ( P＜0.01), as were the ALT and TBA levels ( P＜0.05); however, the ALB content was significantly increased ( P＜0.01), and the differences were statistically significant. Compared with the Sham group, the mRNA expression levels of CK7 and CK19 were significantly increased in the BDL group (1.40±0.42 vs. 43.78±7.56; 1.11±0.51 vs. 363.81±134.84, P＜0.01). The mRNA expression levels of CK7 and CK19 were significantly reduced in the OE group (343.19±81.22 vs. 3.22±2.34; 40.53±14.02 vs. 15.68±9.36, P＜0.01). Conclusion:Overexpression of the Numb gene can inhibit CLF progression in the adult liver, which may become a new target for CLF therapy.

Objective To investigate the effect of polarized bone marrow-derived macrophage (BMDM) transplantation on the progression of CCl 4 -induced liver fibrosis in rats. Methods Rat BMDMs were isolated and induced to differentiate into M1 phenotype (M1-BMDM) by lipopolysaccharide (5 ng/mL) or M2 phenotype (M2-BMDM) by the supernatant of L929 cells. A rat model of liver fibrosis was established by subcutaneous injection of 30% CCl 4 for 6 weeks, and at week 7, the model rats were randomly divided into model control group (M group), M1-BMDM group, and M2-BMDM group and were given a single injection of normal saline, M1-BMDM, and M2-BMDM, respectively, via the caudal vein, and subcutaneous injection of 30% CCl 4 was given until the end of week 9. Related indices were observed, including liver function, liver histopathology, hydroxyproline (Hyp) content in liver tissue, hepatic stellate cell activation, liver fibrosis, and expression of inflammatory cytokines. The continuous data were expressed as mean±standard deviation; an analysis of variance was used for comparison between multiple groups, and the SNK- q test was used for further comparison between two groups. Results Compared with the M group, both M1-BMDM and M2-BMDM significantly inhibited liver inflammation and liver fibrosis progression and significantly reduced serum alanine aminotransferase and aspartate aminotransferase activities ( P < 0.01) and Hyp content in liver tissue ( P < 0.05). M1-BMDM and M2-BMDM significantly inhibited the activation of hepatic stellate cells and significantly reduced the mRNA expression levels of TGF-β, Col1A1, and Col4 (all P < 0.05). Both M1-BMDM and M2-BMDM significantly increased the expression level of CD163 protein in liver tissue ( P < 0.01), and the M2-BMDM group had a significantly higher level than the M1-BMDM group ( P < 0.05); both M1-BMDM and M2-BMDM significantly reduced the mRNA expression levels of MMP-2 and TIMP-1 in liver tissue ( P < 0.05) and significantly increased the mRNA expression level of MMP-13 ( P < 0.01); in addition, M2-BMDM significantly reduced the expression level of CD68 protein in liver tissue ( P < 0.01). Both M1-BMDM and M2-BMDM significantly increased the mRNA expression levels of IL-6 and IL-10 and the protein expression level of albumin in liver tissue (all P < 0.05), and the above indices in the M2-BMDM group were significantly higher than those in the M1-BMDM group (all P < 0.05). Conclusion Both M1-BMDM and M2-BMDM can effectively inhibit the progression of CCl 4 -induced liver fibrosis in rats, possibly by inhibiting the activation of hepatic stellate cells and promoting the activation of anti-inflammatory macrophages. Moreover, M2-BMDM can also inhibit the activation of pro-inflammatory macrophages and thus has a better comprehensive intervention effect than M1-BMDM.

Objective:To analyze the survival benefits and treatment related toxic effects of simultaneous integrated boost intensity-modulated radiotherapy (SIB-RT) for non-operative esophageal squamous cell carcinoma patients.Methods:The data of 2 132 ESCC patients who were not suitable for surgery or rejected operation, and underwent radical radiotherapy from 2002 to 2016 in 10 hospitals of Jing-Jin-Ji Esophageal and Esophagogastric Cancer Radiotherapy Oncology Group (3JECROG) were analyzed. Among them, 518 (24.3%) cases underwent SIB (SIB group) and 1 614 (75.7%) cases did not receive SIB (No-SIB group). The two groups were matched with 1∶2 according to propensity score matching (PSM) method (caliper value=0.02). After PSM, 515 patients in SIB group and 977 patients in No-SIB group were enrolled. Prognosis and treatment related adverse effects of these two groups were compared and the independent prognostic factor were analyzed.Results:The median follow-up time was 61.7 months. Prior to PSM, the 1-, 3-, and 5-years overall survival (OS) rates of SIB group were 72.2%, 42.8%, 35.5%, while of No-SIB group were 74.3%, 41.4%, 31.9%, respectively ( P=0.549). After PSM, the 1-, 3-, and 5-years OS rates of the two groups were 72.5%, 43.4%, 36.4% and 75.3%, 41.7%, 31.6%, respectively ( P=0.690). The univariate survival analysis of samples after PSM showed that the lesion location, length, T stage, N stage, TNM stage, simultaneous chemoradiotherapy, gross tumor volume (GTV) and underwent SIB-RT or not were significantly associated with the prognosis of advanced esophageal carcinoma patients who underwent radical radiotherapy ( P<0.05). Cox model multivariate regression analysis showed lesion location, TNM stage, GTV and simultaneous chemoradiotherapy were independent prognostic factors of advanced esophageal carcinoma patients who underwent radical radiotherapy ( P<0.05). Stratified analysis showed that, in the patients whose GTV volume≤50 cm 3, the median survival time of SIB and No-SIB group was 34.7 and 30.3 months ( P=0.155), respectively. In the patients whose GTV volume>50 cm 3, the median survival time of SIB and No-SIB group was 16.1 and 20.1 months ( P=0.218). The incidence of radiation esophagitis and radiation pneumonitis above Grade 3 in SIB group were 4.3% and 2.5%, significantly lower than 13.1% and 11% of No-SIB group ( P<0.001). Conclusions:The survival benefit of SIB-RT in patients with locally advanced esophageal carcinoma is not inferior to non-SIB-RT, but without more adverse reactions, and shortens the treatment time. SIB-RT can be used as one option of the radical radiotherapy for locally advanced esophageal cancer.

Objective:To analyze the survival benefits and treatment related toxic effects of simultaneous integrated boost intensity-modulated radiotherapy (SIB-RT) for non-operative esophageal squamous cell carcinoma patients.Methods:The data of 2 132 ESCC patients who were not suitable for surgery or rejected operation, and underwent radical radiotherapy from 2002 to 2016 in 10 hospitals of Jing-Jin-Ji Esophageal and Esophagogastric Cancer Radiotherapy Oncology Group (3JECROG) were analyzed. Among them, 518 (24.3%) cases underwent SIB (SIB group) and 1 614 (75.7%) cases did not receive SIB (No-SIB group). The two groups were matched with 1∶2 according to propensity score matching (PSM) method (caliper value=0.02). After PSM, 515 patients in SIB group and 977 patients in No-SIB group were enrolled. Prognosis and treatment related adverse effects of these two groups were compared and the independent prognostic factor were analyzed.Results:The median follow-up time was 61.7 months. Prior to PSM, the 1-, 3-, and 5-years overall survival (OS) rates of SIB group were 72.2%, 42.8%, 35.5%, while of No-SIB group were 74.3%, 41.4%, 31.9%, respectively ( P=0.549). After PSM, the 1-, 3-, and 5-years OS rates of the two groups were 72.5%, 43.4%, 36.4% and 75.3%, 41.7%, 31.6%, respectively ( P=0.690). The univariate survival analysis of samples after PSM showed that the lesion location, length, T stage, N stage, TNM stage, simultaneous chemoradiotherapy, gross tumor volume (GTV) and underwent SIB-RT or not were significantly associated with the prognosis of advanced esophageal carcinoma patients who underwent radical radiotherapy ( P<0.05). Cox model multivariate regression analysis showed lesion location, TNM stage, GTV and simultaneous chemoradiotherapy were independent prognostic factors of advanced esophageal carcinoma patients who underwent radical radiotherapy ( P<0.05). Stratified analysis showed that, in the patients whose GTV volume≤50 cm 3, the median survival time of SIB and No-SIB group was 34.7 and 30.3 months ( P=0.155), respectively. In the patients whose GTV volume>50 cm 3, the median survival time of SIB and No-SIB group was 16.1 and 20.1 months ( P=0.218). The incidence of radiation esophagitis and radiation pneumonitis above Grade 3 in SIB group were 4.3% and 2.5%, significantly lower than 13.1% and 11% of No-SIB group ( P<0.001). Conclusions:The survival benefit of SIB-RT in patients with locally advanced esophageal carcinoma is not inferior to non-SIB-RT, but without more adverse reactions, and shortens the treatment time. SIB-RT can be used as one option of the radical radiotherapy for locally advanced esophageal cancer.

Objective: To evaluate the prognostic factors of T1-2N0M0 esophageal squamous cell carcinoma (ESCC) treated with definitive radiotherapy. Methods: The clinical data of 196 patients with T1-2N0M0 ESCC who were treated with definitive radiotherapy in 10 hospitals were retrospectively analyzed. All sites were members of Jing-Jin-Ji Esophageal and Esophagogastric Cancer Radiotherapy Oncology Group (3JECROG). Radiochemotherapy were applied to 78 patients, while the other 118 patients received radiotherapy only. 96 patients were treated with three-dimensional conformal radiotherapy (3DCRT) and 100 treated with intensity-modulated radiotherapy (IMRT). The median dose of plan target volume(PTV) and gross target volume(GTV) were both 60 Gy. The median follow-up time was 59.2 months. Log rank test and Cox regression analysis were used for univariat and multivariate analysis, respectively. Results: The percentage of normal lung receiving at least 20 Gy (V₂₀) was (18.65±7.20)%, with average dose of (10.81±42.05) Gy. The percentage of normal heart receiving at least 30 Gy (V₃₀) was (14.21±12.28)%. The maximum dose of exposure in spinal cord was (39.65±8.13) Gy. The incidence of radiation pneumonia and radiation esophagitis were 14.80%(29/196) and 65.82%(129/196), respectively. The adverse events were mostly grade 1-2, without grade 4 toxicity. Median overall survival (OS) and progression-free survival (PFS) were 70.1 months and 62.3 months, respectively. The 1-, 3- and 5-year OS rates of all patients were 75.1%、57.4% and 53.2%, respectively. The 1-, 3- and 5-year PFS rates were 75.1%、57.4% and 53.2%, respectively. Multivariate analysis demonstrated that patients′age (HR=1.023, P=0.038) and tumor diameter (HR=1.243, P=0.028)were the independent prognostic factors for OS, while tumor volume were the independent prognostic factor for PFS. Conclusions Definitive radiotherapy is a promising therapeutic method in patients with T1-2N0M0 ESCC. Patients′ age, tumor diameter and tumor volume may impact patients′ prognosis.

With the outbreak of COVID-19 around the globe, the epidemic prevention and control in China will take a long-term trend. As the main treatment of gynecological malignant tumor, rational application of radiotherapy bring patients with both "epidemic prevention" and "anti-tumor" benefits. This recommendation combined the domestic epidemic prevention guidelines, foreign literature related to epidemic prevention and gynecological tumor treatment, and the experiences of Peking Union Medical College Hospital during the prophase of epidemic period, aiming to provide guidance for the clinical work for radiotherapy and gynecological departments during COVID-19 crisis from the perspectives of gynecological radiotherapy principles, clinical trials, MDT and academic activities.