Background and purpose:Despite Radiation-induced lymphopenia has been associated with poor survival outcomes in certain solid tumors,there is limited evidence for small cell lung cancer(SCLC).The purpose of this study was to investigate whether the absolute lymphocyte count before and after radiotherapy could predict the clinical outcomes for limited-stage SCLC(LS-SCLC)patients.Methods:This was a single-center,retrospective cohort study.A retrospective analysis of patients evaluated at Fudan University Shanghai Cancer Center from January 2007 to December 2017 was conducted.Inclusion criteria:⑴ pathologically confirmed small-cell lung cancer;⑵ limited-stage disease defined by positron emission tomography and computed tomography(PET/CT)and contrast-enhanced brain magnetic resonance imaging(MRI)[American Joint Committee on Cancer(AJCC)8th edition TNM stage M0];⑶ receipt of definitive chemoradiotherapy;⑷ availability of complete blood counts before,during and within 1 month after radiotherapy;⑸ complete survival,relapse,and last-follow-up information retrievable.Exclusion criteria:⑴ distant metastasis at baseline(AJCC 8th edition TNM stage M1,including any distant nodal,visceral,or bone-marrow involvement);⑵ total radiotherapy dose＜50 Gy[calculated as an equivalent biological dose at 2 Gy/fraction,i.e.,a biological effective dose(BED)＜40 Gy];⑶ incomplete laboratory data at any scheduled time point;⑷ inability to ascertain survival or relapse status or insufficient follow-up records.The study protocol was approved by the ethics committee of Fudan University Shanghai Cancer Center(approval number:2303271-15),and the requirement for informed consent was waived.Clinical data extracted comprised age,sex,Eastern Cooperative Oncology Group performance status(ECOG PS)score,smoking history,TNM stage,chemotherapy regimen and number of cycles,radiotherapy dose and fractionation schedule,use of concurrent chemoradiotherapy and administration of prophylactic cranial irradiation(PCI).Laboratory data comprised serial absolute lymphocyte counts obtained within 1 month before,during and after radiotherapy;lymphopenia was graded according to the Common Terminology Criteria for Adverse Events(CTCAE)version 4.0.Progression-free survival(PFS)and overall survival(OS)were estimated using the Kaplan-Meier method and compared with the log-rank test.Results:A total of 170 patients were included.The median age of the patients was 57 years,with 77.6%being male.The median radiation therapy dose was 60 Gy(range:45-66 Gy).For the entire cohort,the median PFS was 22.0 months,the 5-year PFS rate was 31.3%,and the 10-year PFS rate was 19.8%.The median OS was 38.0 months,the 5-year OS rate was 37.5%,and the 10-year OS rate was 24.2%.Before radiation therapy,14 patients(8.2%)had grade 1-2 lymphocytopenia.During radiation therapy,the number of patients with grade 1,2,3 and 4 lymphocytopenia was 7(4.1%),22(12.9%),111(65.3%),and 24(14.1%),respectively.One month after radiation therapy,the number of patients with grade 1,2,3 and 4 lymphocytopenia was 36(21.2%),36(21.2%),11(6.5%)and 1(0.6%),respectively.There were no significant differences in PFS and OS among patients with different grades of lymphocytopenia before,during,or after radiation therapy.Conclusion:Before immunotherapy,radiotherapy-induced lymphopenia did not appear to affect the prognosis of patients with LS-SCLC.

Background and purpose:Despite Radiation-induced lymphopenia has been associated with poor survival outcomes in certain solid tumors,there is limited evidence for small cell lung cancer(SCLC).The purpose of this study was to investigate whether the absolute lymphocyte count before and after radiotherapy could predict the clinical outcomes for limited-stage SCLC(LS-SCLC)patients.Methods:This was a single-center,retrospective cohort study.A retrospective analysis of patients evaluated at Fudan University Shanghai Cancer Center from January 2007 to December 2017 was conducted.Inclusion criteria:⑴ pathologically confirmed small-cell lung cancer;⑵ limited-stage disease defined by positron emission tomography and computed tomography(PET/CT)and contrast-enhanced brain magnetic resonance imaging(MRI)[American Joint Committee on Cancer(AJCC)8th edition TNM stage M0];⑶ receipt of definitive chemoradiotherapy;⑷ availability of complete blood counts before,during and within 1 month after radiotherapy;⑸ complete survival,relapse,and last-follow-up information retrievable.Exclusion criteria:⑴ distant metastasis at baseline(AJCC 8th edition TNM stage M1,including any distant nodal,visceral,or bone-marrow involvement);⑵ total radiotherapy dose＜50 Gy[calculated as an equivalent biological dose at 2 Gy/fraction,i.e.,a biological effective dose(BED)＜40 Gy];⑶ incomplete laboratory data at any scheduled time point;⑷ inability to ascertain survival or relapse status or insufficient follow-up records.The study protocol was approved by the ethics committee of Fudan University Shanghai Cancer Center(approval number:2303271-15),and the requirement for informed consent was waived.Clinical data extracted comprised age,sex,Eastern Cooperative Oncology Group performance status(ECOG PS)score,smoking history,TNM stage,chemotherapy regimen and number of cycles,radiotherapy dose and fractionation schedule,use of concurrent chemoradiotherapy and administration of prophylactic cranial irradiation(PCI).Laboratory data comprised serial absolute lymphocyte counts obtained within 1 month before,during and after radiotherapy;lymphopenia was graded according to the Common Terminology Criteria for Adverse Events(CTCAE)version 4.0.Progression-free survival(PFS)and overall survival(OS)were estimated using the Kaplan-Meier method and compared with the log-rank test.Results:A total of 170 patients were included.The median age of the patients was 57 years,with 77.6%being male.The median radiation therapy dose was 60 Gy(range:45-66 Gy).For the entire cohort,the median PFS was 22.0 months,the 5-year PFS rate was 31.3%,and the 10-year PFS rate was 19.8%.The median OS was 38.0 months,the 5-year OS rate was 37.5%,and the 10-year OS rate was 24.2%.Before radiation therapy,14 patients(8.2%)had grade 1-2 lymphocytopenia.During radiation therapy,the number of patients with grade 1,2,3 and 4 lymphocytopenia was 7(4.1%),22(12.9%),111(65.3%),and 24(14.1%),respectively.One month after radiation therapy,the number of patients with grade 1,2,3 and 4 lymphocytopenia was 36(21.2%),36(21.2%),11(6.5%)and 1(0.6%),respectively.There were no significant differences in PFS and OS among patients with different grades of lymphocytopenia before,during,or after radiation therapy.Conclusion:Before immunotherapy,radiotherapy-induced lymphopenia did not appear to affect the prognosis of patients with LS-SCLC.

Anterolateral thigh flap (ALTF) has been widely applied in reconstruction of soft tissue defects due to its abundant blood supply, excellent flexibility and flexible in design. With continuous development in digital technologies, the application of flap surgery runs through the key stages in preoperative planning, intraoperative navigation and postoperative monitoring. Before the surgery, individualised surgical planning through the precise digital technology can optimise the design of flaps and improve the accuracy of surgery. During the surgery, accurate dissection and effective protection of the perforating vessel can be achieved by the combination with navigation technology to reduce the intraoperative risks. After the surgery, blood supply of a flap can be dynamically evaluated by various advanced monitoring methods, which may help to identify a vascular compromise in time, prevent complications and significantly improve the survival of the flap. Although digital technology has been widely applied in all stages of flap surgery, the specific application of digital technology in ALTF has not yet been reported in detail. Therefore, based on the research progresses at home and abroad, this paper systematically reviews the clinical application, preoperative planning, intraoperative navigation and postoperative monitoring of various digital technologies in the surgery of ALTF, thereby to provide theoretical support and references for microsurgical practices.

Objective:To investigate the effects of dual vascularized tissue-engineered bone constructed by vascular bundles and endothelial progenitor cells (EPCs) on repair of large bone defects and vascular regeneration.Methods:EPCs were seeded on the demineralized bone matrix (DBM) scaffolds and cultured for 6 days. The attachment and morphology of EPCs on DBM scaffolds were observed by electron microscopy. Next, the radial artery was implanted into a vascular groove opened inside the DBM-EPCs composite scaffolds. Finally, models of a large segmental bone defect were constructed using the radii from 18 New Zealand white rabbits. The rabbits were randomly divided into 4 groups using a simple random sampling method: DBM group, DBM+EPCs group, DBM+vascular bundle group, and DBM+EPCs+vascular bundle group. The DBM group and DBM+EPCs group shared the same rabbits so that transplantations were conducted into the left and right forearms respectively; the DBM+vascular bundle group and DBM+EPCs+vascular bundle group also shared the same rabbits so that transplantations were conducted into the left and right forearms respectively. Consequently, there were 9 experimental sites in each group. X-ray examination and gross morphological observation were performed to evaluate the bone regeneration in the experimental rabbits in each group at 4, 8, and 12 weeks after surgery, and CD31 immunofluorescence staining was used to evaluate the vascular regeneration. Micro-CT was used to analyze bone tissue parameters and reconstruct the three-dimensional structures of the defects site at 12 weeks after surgery.Results:Compared with the DBM, DBM+EPCs and DBM+vascular bundle groups, the DBM+EPCs+vascular bundle group showed new bone tissue crawling on the scaffold surface at 4 weeks after surgery, almost complete healing of the bone defect area at 8 weeks, and forming of a complete and dense bone bridge and appearance of a bone marrow cavity at 12 weeks. Micro-CT data at 12 weeks after surgery showed regular arrangement of the trabeculae, significantly improved mineralization, and increased thickness of the bone cortex in the DBM+EPCs+vascular bundle group. Additionally, in the DBM+EPCs+vascular bundle group, the number of microvessels was significantly higher than that in the other groups at 4, 8, 12 weeks after surgery ( P<0.05), and the angiogenesis and bone tissue regeneration were particularly prominent at 12 weeks after surgery. The number of CD31 cells in the DBM+EPCs+vascular bundle group increased significantly more than that in the DBM, DBM+EPCs and DBM+vascular bundle groups ( P<0.05). Conclusion:As the dual vascularized tissue-engineered bone constructed by vascular bundles and EPCs can significantly promote bone tissue regeneration and angiogenesis, it may be a potential therapeutic strategy for repair of large bone defects.

Objective:To investigate the effect of mild moxibustion on transient receptor potential vanilloid type 1(TRPV1)channel expression in primary dysmenorrhea(PD)rats and explore its mechanism in alleviating central pain sensitization.Methods:Thirty-two female non-pregnant Wistar rats were randomized into a blank group,a model group,a mild moxibustion group,and a capsazepine group,with 8 rats in each group.Except for the blank group,the other three groups used estradiol benzoate,ice-water bath,and oxytocin to establish the rat PD model of cold-dampness stagnation pattern.The interventions began on day 1 of modeling,once a day,and lasted 10 d.The mild moxibustion group received mild moxibustion at Shenque(CV8)and Guanyuan(CV4),20 min/time;in the capsazepine group,capsazepine was injected at a dose of 2 mg/(kg·bw).The abdominal pain threshold was measured 10-30 min after oxytocin injection on day 11;enzyme-linked immunosorbent assay was used to detect serum prostaglandin F2α(PGF2α)level;the expression of TRPV1,cluster of differentiation 11B(CD11B),and proto-oncogene c-Fos in the spinal dorsal horn and hypothalamus was detected by immunofluorescence and Western blotting.Results:Compared to the blank group,the model group showed a decreased pain threshold(P<0.05)and an increased serum PGF2α level with elevated TRPV1,CD11B,and c-Fos protein expression in the spinal dorsal horn and hypothalamus(P<0.05).Compared to the model group,both the mild moxibustion group and capsazepine group showed significantly increased pain thresholds(P<0.05),along with decreased serum PGF2α levels and reduced protein expression levels of TRPV1,CD11B,and c-Fos in the spinal dorsal horn and hypothalamus(P<0.05).Rat pain threshold in the capsazepine group was higher than that in the mild moxibustion group(P<0.05).Serum PGF2α level,the expression levels of CD11B and c-Fos proteins in the spinal dorsal horn,as well as TRPV1,CD11B,and c-Fos proteins in the hypothalamus of the capsazepine group were lower than those in the mild moxibustion group(P<0.05).Conclusion:Mild moxibustion at Shenque(CV8)and Guanyuan(CV4)may alleviate the central pain sensitization in PD rats by down-regulating TRPV1 channel expression in the spinal dorsal horn and hypothalamus,thus playing an analgesic effect.

Objective:To investigate the effect of mild moxibustion on transient receptor potential vanilloid type 1(TRPV1)channel expression in primary dysmenorrhea(PD)rats and explore its mechanism in alleviating central pain sensitization.Methods:Thirty-two female non-pregnant Wistar rats were randomized into a blank group,a model group,a mild moxibustion group,and a capsazepine group,with 8 rats in each group.Except for the blank group,the other three groups used estradiol benzoate,ice-water bath,and oxytocin to establish the rat PD model of cold-dampness stagnation pattern.The interventions began on day 1 of modeling,once a day,and lasted 10 d.The mild moxibustion group received mild moxibustion at Shenque(CV8)and Guanyuan(CV4),20 min/time;in the capsazepine group,capsazepine was injected at a dose of 2 mg/(kg·bw).The abdominal pain threshold was measured 10-30 min after oxytocin injection on day 11;enzyme-linked immunosorbent assay was used to detect serum prostaglandin F2α(PGF2α)level;the expression of TRPV1,cluster of differentiation 11B(CD11B),and proto-oncogene c-Fos in the spinal dorsal horn and hypothalamus was detected by immunofluorescence and Western blotting.Results:Compared to the blank group,the model group showed a decreased pain threshold(P<0.05)and an increased serum PGF2α level with elevated TRPV1,CD11B,and c-Fos protein expression in the spinal dorsal horn and hypothalamus(P<0.05).Compared to the model group,both the mild moxibustion group and capsazepine group showed significantly increased pain thresholds(P<0.05),along with decreased serum PGF2α levels and reduced protein expression levels of TRPV1,CD11B,and c-Fos in the spinal dorsal horn and hypothalamus(P<0.05).Rat pain threshold in the capsazepine group was higher than that in the mild moxibustion group(P<0.05).Serum PGF2α level,the expression levels of CD11B and c-Fos proteins in the spinal dorsal horn,as well as TRPV1,CD11B,and c-Fos proteins in the hypothalamus of the capsazepine group were lower than those in the mild moxibustion group(P<0.05).Conclusion:Mild moxibustion at Shenque(CV8)and Guanyuan(CV4)may alleviate the central pain sensitization in PD rats by down-regulating TRPV1 channel expression in the spinal dorsal horn and hypothalamus,thus playing an analgesic effect.

Anterolateral thigh flap (ALTF) has been widely applied in reconstruction of soft tissue defects due to its abundant blood supply, excellent flexibility and flexible in design. With continuous development in digital technologies, the application of flap surgery runs through the key stages in preoperative planning, intraoperative navigation and postoperative monitoring. Before the surgery, individualised surgical planning through the precise digital technology can optimise the design of flaps and improve the accuracy of surgery. During the surgery, accurate dissection and effective protection of the perforating vessel can be achieved by the combination with navigation technology to reduce the intraoperative risks. After the surgery, blood supply of a flap can be dynamically evaluated by various advanced monitoring methods, which may help to identify a vascular compromise in time, prevent complications and significantly improve the survival of the flap. Although digital technology has been widely applied in all stages of flap surgery, the specific application of digital technology in ALTF has not yet been reported in detail. Therefore, based on the research progresses at home and abroad, this paper systematically reviews the clinical application, preoperative planning, intraoperative navigation and postoperative monitoring of various digital technologies in the surgery of ALTF, thereby to provide theoretical support and references for microsurgical practices.

Objective:To investigate the effects of dual vascularized tissue-engineered bone constructed by vascular bundles and endothelial progenitor cells (EPCs) on repair of large bone defects and vascular regeneration.Methods:EPCs were seeded on the demineralized bone matrix (DBM) scaffolds and cultured for 6 days. The attachment and morphology of EPCs on DBM scaffolds were observed by electron microscopy. Next, the radial artery was implanted into a vascular groove opened inside the DBM-EPCs composite scaffolds. Finally, models of a large segmental bone defect were constructed using the radii from 18 New Zealand white rabbits. The rabbits were randomly divided into 4 groups using a simple random sampling method: DBM group, DBM+EPCs group, DBM+vascular bundle group, and DBM+EPCs+vascular bundle group. The DBM group and DBM+EPCs group shared the same rabbits so that transplantations were conducted into the left and right forearms respectively; the DBM+vascular bundle group and DBM+EPCs+vascular bundle group also shared the same rabbits so that transplantations were conducted into the left and right forearms respectively. Consequently, there were 9 experimental sites in each group. X-ray examination and gross morphological observation were performed to evaluate the bone regeneration in the experimental rabbits in each group at 4, 8, and 12 weeks after surgery, and CD31 immunofluorescence staining was used to evaluate the vascular regeneration. Micro-CT was used to analyze bone tissue parameters and reconstruct the three-dimensional structures of the defects site at 12 weeks after surgery.Results:Compared with the DBM, DBM+EPCs and DBM+vascular bundle groups, the DBM+EPCs+vascular bundle group showed new bone tissue crawling on the scaffold surface at 4 weeks after surgery, almost complete healing of the bone defect area at 8 weeks, and forming of a complete and dense bone bridge and appearance of a bone marrow cavity at 12 weeks. Micro-CT data at 12 weeks after surgery showed regular arrangement of the trabeculae, significantly improved mineralization, and increased thickness of the bone cortex in the DBM+EPCs+vascular bundle group. Additionally, in the DBM+EPCs+vascular bundle group, the number of microvessels was significantly higher than that in the other groups at 4, 8, 12 weeks after surgery ( P<0.05), and the angiogenesis and bone tissue regeneration were particularly prominent at 12 weeks after surgery. The number of CD31 cells in the DBM+EPCs+vascular bundle group increased significantly more than that in the DBM, DBM+EPCs and DBM+vascular bundle groups ( P<0.05). Conclusion:As the dual vascularized tissue-engineered bone constructed by vascular bundles and EPCs can significantly promote bone tissue regeneration and angiogenesis, it may be a potential therapeutic strategy for repair of large bone defects.

OBJECTIVE To establish the cells stably co-expressing α1A-adrenergic receptor(α1A-AR)and enhanced green fluorescent protein(EGFP)tagged nuclear factor of activated T cells 2(NFAT2)(EGFP-NFAT2)in U2OS cells.METHODS ① The pcDNA3.1-α1-AR-3×FLAG recombinant plasmid was transfected into U2OS-EGFP-NFAT2 cells.The transfected cells were selected by hygromycin B(Hygro-B,200 mg·L-1),and screened by EGFP-NFAT2 nuclear translocation assay after α1A-AR agonist norepinephrine(NE)treatment of 30 min.② The mRNA and protein expression levels of α1A-AR in the selected U2OS-EGFP-NFAT2-α1A-AR cells were examined by real-time quantitative PCR(RT-qPCR)and Western blotting.③ U2OS-EGFP-NFAT2-α1A-AR cells were treated with NE(10-8-10-5 mol·L-1)or dexmedetomidine(DMED,10-8.8-10-5 mol·L-1),respectively,for 30 min.EGFP-NFAT2 nuclear translo-cation was detected by high throughout screening assay.④ The U2OS-EGFP-NFAT2-α1A-AR cells were divided into the solvent control group,α1-AR antagonist naftopidill(1 μmol·L-1)group,NE(1 μmol·L-1)group and naftopidill+NE(co-incubation with naftopidill 1 μmol·L-1 and NE 1 μmol·L-1)group,α2-AR antagonist atipamezole(ATI,0.1 μmol·L-1)group,α 2-AR agonist DMED(0.1 μmol·L-1)group,and ATI+DMED(co-incubation with ATI 1 μmol·L-1 and DMED 0.1 μmol·L-1)group.The drug incubation time was 30 min.EGFP-NFAT2 nuclear translocation was abserved via a high throughout screening system to validate the α1A-AR function in U2OS-EGFP-NFAT2-α1A-AR cells.RESULTS ① There were 58 cell strains expressing α1A-AR in U2OS-EGFP-NFAT2 cells by EGFP-NFAT2 nuclear translocation assay.Among these cells,cells No 50 had the highest nuclear translocation function.The α1A-AR mRNA expression of cells No 50 in 5-20 generations were detected by RT-qPCR and were about 500-800 times that of U2OS-EGFP-NFAT2 cells.② The protein band of α1A-AR was also detected in cells No 50,but no band of α1A-AR was detected in U2OS-EGFP-NFAT2 cells by Western blotting.③ NE and DMED increased the relative translocation nuclear index in U2OS-EGFP-NFAT2-α1A-AR cells with ED50 5.94×10-7 and 6.15×10-8 mol·L-1,respectively.④ EGFP-NFAT2 nuclear translocation was significant in U2OS-EGFP-NFAT2-α1A-AR cells after NE addition compared with the solvent control or the naftopidill groups(P<0.01).The EGFP-NFAT2 nuclear translocation in the naftopidill+NE group was significantly decreased compared with the NE group(P<0.01).DMED significantly increased the EGFP-NFAT2 nuclear translocation compared with solvent control or the ATI groups(P<0.01).The EGFP-NFAT2 nuclear translocation in the ATI+DMED group was similar to that of the DMED group.The EGFP-NFAT2 nuclear translocation in the naftopidill+DMED group was decreased significantly compared with the DMED group(P<0.01).CONCLUSION U2OS-EGFP-NFAT2-α1A-AR cells stably co-exrepssing α1A-AR and EGFP-NFAT2 are established,which can be used for high throughout screening of biased chemicals and studies on the mechanism of α1A-AR.

Objective To explore the mechanism of the treatment of primary dysmenorrhea(PD)by mild moxibustion on"Shenque"and"Guanyuan"acupoints based on the regulation of cAMP-PKA signaling pathway on TRPV1.Methods Totally 32 female non-pregnant Wistar rats were randomly divided into blank group,model group,mild moxibustion group and capsazepine group,with 8 rats in each group.Except for the blank group,the other groups all used estradiol benzoate intraperitoneal injection combined with ice water bath to establish a PD cold-dampness stagnation syndrome rat model.Intervention began on the first day of modeling,the mild moxibustion group selects"Shenque"and"Guanyuan"for mild moxibustion,20 min per time,the capsazepine group was injected capsazepine 2 mg/kg,once a day for 10 consecutive days.ELISA was used to detect uterine PGF2α and cAMP content,immunofluorescence staining was used to detect TRPV1 expression in uterine tissue,Western blot was used to detect PKA,p-PKA and TRPV1 protein expression.Results Compared with the blank group,the latency period of body twisting in the model group rats decreased,and the body twisting score increased(P<0.01);the contents of PGF2α and cAMP in uterine tissue increased(P<0.01),and the expressions of TRPV1 and p-PKA proteins increased(P<0.01).Compared with the model group,the mild moxibustion group and capsazepine group showed an increase in the latency period of body twisting and a decrease in the body twisting score(P<0.01);the content of PGF2α and cAMP in uterine tissue decreased(P<0.01),and the expressions of TRPV1 and p-PKA proteins decreased(P<0.05,P<0.01).Compared with the mild moxibustion group,the capsazepine group showed an increase in the latency period of body twisting and a decrease in the body twisting score(P<0.01);the contents of PGF2α and cAMP in uterine tissue decreased(P<0.05,P<0.01),and the expressions of TRPV1 protein decreased(P<0.05).Conclusion Mild moxibustion at"Shenque"and"Guanyuan"acupoints has obvious analgesic effect on PD rats,and its mechanism may be related to the regulation of uterine cAMP-PKA signaling pathway mediated TRPV1 protein expression.