Objective: To investigate the effects of PssL-NAC reactive oxygen species (ROS)-responsive nanoparticles on intracellular ROS production, inflammatory factor levels, collagen production, cell function and Toll-like receptor 4 (TLR4), NF-κB nuclear factor-κB (p65) pathway protein expression in human gingival fibroblasts (HGFs) induced by Porphyromonas gingivalis-lipopolysaccharide (P.g-LPS). Methods: This study was reviewed and approved by the ethics committee. PssL-NAC microspheres containing oil soluble antioxidant N-acetylcysteine (NAC) were obtained by connecting the hydrophobic end of polycaprolactone (PCL) and the hydrophilic end of polyethylene glycol (PEG) via thioketal (TK) bonds in response to ROS, and self loading in the aqueous and oil phases. After preparation of the PssL-NAC microspheres and aqueous NAC solution, successful synthesis of the nanoparticles was verified by transmission electron microscopy. Then, HGFs were exposed to P.g-LPS (0, 5, or 10 μg/mL), P.g-LPS (0, 5, or 10 μg/mL)+NAC, and P.g-LPS (0, 5, or 10 μg/mL)+PssL-NAC, and the ROS levels in the different groups were observed under confocal microscopy to determine the concentration of P.g-LPS for use in subsequent experiments. The groups were as follows: control group (no treatment), P.g-LPS group (HGFs treated with P.g-LPS), NAC group (HGFs treated with P.g-LPS and NAC), and PssL-NAC group (HGFs treated with P.g-LPS and PssL-NAC). Cell counting kit-8 (CCK-8) assays verified the biosafety of PssL-NAC. The ROS levels in the different groups were detected by DCFH-DA probes and observed via confocal microscopy. Real-time qPCR (RT-qPCR) was used to monitor the gene expression levels of the intracellular inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), collagen 1 (COL1) and collagen 3 (COL3). The effect of PssL-NAC on the migration of HGFs was observed via the scratch test. The protein expression of TLR4-NF-κB, and phosphorylated p65 (p-p65) in the TLR4-NF-κB pathway was evaluated by Western blot. Results: PssL-NAC had no significant effect on HGF proliferation (P>0.05). At elevated P.g-LPS concentrations, PssL-NAC maintained intracellular ROS levels approximately twice those in the control group (P<0.001). PssL-NAC significantly decreased P.g-LPS-induced IL-6 (P<0.001) and TNF-α (P<0.001) gene expression and increased COL1 gene expression (P<0.001). After P.g-LPS stimulation, PssL-NAC restored cell migration to the control level (P>0.05) and decreased the protein expression of TLR4 (P<0.001), p65 (P = 0.006), and p-p65 (P = 0.017) in the TLR4-NF-κB pathway. Conclusion PssL-NAC maintains the appropriate intracellular ROS concentration, alleviates P.g-LPS-induced inflammation in HGFs through the TLR4-NF-κB pathway, and restores the cell functions of collagen production and migration in an inflammatory environment.

[摘 要] 嵌合抗原受体基因修饰T（CAR-T）细胞免疫治疗被认为是最有前景的肿瘤治疗方法之一，效应CAR-T细胞的数量是决定CAR-T细胞疗法治疗效果的关键因素。CAR-T细胞的体外扩增耗时耗力，回输体内后，CAR-T细胞大量耗竭且难以浸润实体瘤，导致能有效抑制实体瘤的CAR-T细胞数量大幅下降。目前，CAR-T细胞的扩增方法在提高扩增特异性和治疗安全性等方面均存在问题，为CAR-T细胞疗法的临床转化造成困难。近年来，新型免疫激动剂及其下游信号的发现为CAR-T细胞扩增方案提供了更多选择，免疫激动剂给药方式的更新迭代进一步提高了其在体内扩增CAR-T细胞的安全性。本文分析了目前扩增CAR-T细胞面临的挑战，系统阐述了近年来在体内外扩增CAR-T细胞的新策略，为CAR-T细胞疗法的疗效和产能优化提供了新思路。

Objective To investigate the predictive value of preoperative blood inflammatory markers for the recurrence risk in patients with basal cell carcinoma(BCC).Methods A total of 225 patients with BCC were divided into the high-risk recurrence group(155 cases)and the low-risk recurrence group(70 cases).General information and preoperative hematological indicators were collected in the two groups of patients.The neutrophil-to-lymphocyte ratio(NLR),lymphocyte-to-monocyte ratio(LMR),systemic inflammation marker(SIM)and platelet-to-lymphocyte ratio(PLR)were calculated.Receiver operating characteristic(ROC)curves were used to determine the predictive value of hematological markers with statistically significant differences between the two groups for BCC recurrence and to establish optimal cutoff values.Univariate and multivariate Logistic regression analyses were conducted to identify factors influencing BCC recurrence.A multivariate Logistic regression model was established to predict the recurrence risk of BCC.Area under the curve(AUC)and the Hosmer-Lemeshow test were used to evaluate the prediction efficiency and goodness-of-fit of the model.Results ROC analysis identified that optimal cutoff values for LMR and SIM were 5.12 and 0.86,respectively.Univariate Logistic regression analysis showed that LMR,SIM,ulceration at the primary tumor site,UV exposure and tumor maximum diameter were factors influencing BCC recurrence.Multivariate Logistic regression revealed that SIM≥0.86,tumor maximum diameter≥2.0 cm and UV exposure were risk factors for BCC recurrence,while LMR≥5.12 had a protective effect.The Logistic prediction model for BCC recurrence risk was Logit(P)=-1.598-1.517×LMR+1.323×SIM+2.406×UV exposure+3.465×tumor maximum diameter,with good model fit(P=0.725)and an AUC of 0.869(95%CI:0.822-0.917)for predicting BCC recurrence risk.Conclusion Monitoring preoperative LMR and SIM levels can assist in assessing the risk of recurrence in BCC patients and provide important guidance for clinical decision-making.

Purpose/Significance To construct a Chinese medical knowledge Q&A corpus dataset as a standardized evaluation bench-mark for large language models(LLMs)in the medical domain,so as to improve the accuracy and efficiency of LLMs in handling Chinese medical questions.Method/Process Chinese medical paper knowledge,medical terminology explanations and supplementary questions are acquired from the Chinese medical licensing examination,and open-source Chinese medical Q&A datasets are encompassed in the developed Q&A datasets.Result/Conclusion The Chinese medical knowledge Q&A corpus datasets enrich the sources of existing datasets and promote the objective and comprehensive quantitative evaluation of large models in the medical field.In the near future,additional data such as electronic medical records and those from online health communities will be used to strengthen the support of artificial intelli-gence for the Healthy China strategy.

Objective : To investigate the effects of angiopoietin 4 (ANGPT4) on the odontogenic differentiation of human dental pulp stem cells. Methods : This study has been reviewed and approved by the Ethics Committee, and informed consent has been obtained from patients. Human premolars were fixed, decalcified, dehydrated, embedded, and sectioned. Immunofluorescence staining was used to observe the expression and localization of ANGPT4. Human dental pulp stem cells (hDPSCs) were isolated and cultured in vitro. The growth state and morphology of hDPSCs were observed under an inverted phase contrast microscope. The expression of cell surface-related molecular markers was detected by flow cytometry. Alkaline phosphatase and alizarin red S staining were used to detect the odontogenic differentiation potential of hDPSCs. Oil-red O staining was used to detect the adipogenic differentiation potential of hDPSCs. RNA was extracted from hDPSCs at different time points after odontogenic induction, and RT-qPCR was used to analyze the mRNA expression of ANGPT4 and odontogenic-related genes during the odontogenic differentiation of hDPSCs in vitro. siRNA gene silencing technology was used to silence the expression of ANGPT4 in hDPSCs, and the silencing efficiency was detected by RT-qPCR and Western Blot. After silencing ANGPT4 in hDPSCs for 24 h, odontogenic induction was performed. Alkaline phosphatase and alizarin red S staining were performed on the 7th and 14th of induction to detect the odontogenic differentiation ability of hDPSCs after silencing ANGPT4 Results : Immunofluorescence staining of human premolars showed that ANGPT4 was expressed in odontoblasts and sub-odontoblastic cell-rich zone. hDPSCs were in good condition after 14 days of isolation and culture. Under the microscope, multiple cell colonies were observed, and the cell morphology was uniform and long spindle-shaped. The results of flow cytometry showed that hDPSCs expressed mesenchymal stem cell markers CD105 (90.42%) and CD90 (97.15%), but did not express hematopoietic cell markers CD45 (0.01%) and CD34 (0.08%). After odontogenic and adipogenic induction of hDPSCs, alkaline phosphatase staining, alizarin red S staining and oil red O staining were positive. The results of RT-qPCR after the odontogenic induction of hDPSCs showed that ANGPT4 was highly expressed on the 5th, 7th, 11th and 14th days of differentiation of hDPSCs (P<0.05), with the highest expression level on the 5th day. After hDPSCs were transfected with si-ANGPT4, the expression of ANGPT4 mRNA and protein was significantly down-regulated (P<0.05). The results of alkaline phosphatase staining showed that ALP staining intensity and area in the si-ANGPT4 group were significantly lower than those in the negative control. Alizarin red S staining showed that the formation of calcium nodules in the si-ANGPT4 group was significantly lower than that in the negative control. Conclusion ANGPT4 was expressed in odontoblasts and sub-odontoblastic cell-rich zone of human premolars. ANGPT4 may be a factor to promote the odontogenic differentiation of hDPSCs.

Objective: To investigate bone mineral density of college students with masked obesity, and to provide theoretical basis for bone density improvement and osteoporosis prevention in college students. Methods: Participants were enrolled from universities and colleges. A total of 518 low-weight or normal-weight students were included and were classified according to the criteria of masked obesity, dual-energy X-ray test was used to detect the percentage of body fat and bone mineral density of college students in each group. The differences in bone mineral density between masked obesity and non-masked obesity groups of different genders were compared, and the correlation between body mass index, body fat percentage and bone mineral density was analyzed. Results: Among the low-weight male college students, the whole body bone mineral density, t-value and Z-value of masked obesity group were lower than those of the nonmasked obesity group [(1.82±0.04,2.01±0.22)g/cm 2; (-1.10±0.24,-0.02±0.15); (-0.94±0.64,-0.01±0.43)(P<0.01)]. Among low-weight male college students, bone mineral density of the upper limbs, thighs, ribs, and pelvis of the masked obesity group was lower than that of the non-masked obesity group. Among low-weight female college students, body bone mineral density of masked obesity group was lower than that of the non-masked obesity group [(1.13±0.48,1.31±0.29)g/cm 2; (P<0.05)]. Among low-weight female college students, bone mineral density of head, upper limbs, thighs, and trunk of masked obesity group was lower than that of the nonmasked obesity group. In male college students, body mass index was positively correlated with bone mineral density, T-value and Z-value (r=0.69, 0.68, 0.61, P＜0.01) while body fat percentage was negatively correlated with bone mineral density, T-value and Z-value (r=-0.52, -0.51, -0.49, P＜0.01). In female college students, body mass index was positively correlated with T-value and Z-value (r=0.46, 0.26, P＜0.01), and body fat percentage was negatively correlated with T-value and Z-value (r=-0.22, -0.23, P＜0.01). Conclusion Compared with normal-weight college students, depletion of bone mineral density among masked obesity students is observed. It is of great significance to pay attention to bone mineral density of college students with masked obesity and implement intervention to prevent osteoporosis timely.

Objective:To study the expression level of CD73 in cutaneous melanoma and its clinical significance, and to explore its role and molecular mechanism in the occurrence and development of melanoma.Methods:Expression level of the gene NT5E in melanoma was analyzed according to RNA sequencing data from the CCLE database. CD73 expression level was tested in 145 melanoma tissues and corresponding para-tumor tissues by immunohistochemistry tissue microarray. The samples came from 78 male and 67 female patients who received surgical resection and were pathologically diagnosed as melanoma in the department of plastic surgery in Zhongshan Hospital from January of 2008 to December of 2017. The relationship between CD73 expression level and clinicopathological features as well as prognosis was analyzed. Melanoma cell line was transfected with CD73 gene overexpression lentivirus and negative control vectors, respectively. The transfection efficiency was verified by qRT-PCR and Western blot. CCK-8, wound healing and Transwell assays were used to evaluate the proliferation, migration and invasion abilities of the overexpression group and control group. Subcutaneous xenograft tumor models were established in nude mice to assess the tumorigenicity of the two groups. Transcriptome sequencing, GO and KEGG analyses were performed. The expression levels of pAKT and pGSK3β were detected by Western blot. The measurement data were expressed as Mean ±SD. Independent sample t-test was used for comparison between the two groups, and One-way ANOVA was employed for multi-group comparisons. The chi-square test was used for the categorical variables. P<0.05 was considered as statistically significant difference. Results:The expression of CD73 in melanoma was higher than that in para-tumor tissues ( P< 0.05). Higher expression level of CD73 was associated with higher Clark grade ( P= 0.014) and clinical stage ( P= 0.040). The overall survival and disease-free survival of patients with high-CD73-expression were significantly lower than those of patients with low-CD73-expression. CD73 expression, Clark grade and clinical stage are independent risk factors for the poor prognosis of patients with melanoma. Overexpression of CD73 significantly enhanced the proliferation, migration and invasion of melanoma cells in vitro and the tumorigenicity in vivo( P< 0.05). Transcriptome sequencing showed that CD73 was involved in several tumor related signaling pathways, among which the expression of pAKT and pGSK3β was elevated( P< 0.05). Conclusions:CD73 promotes the proliferation, migration and invasion of melanoma cells in vitro and the tumorigenicity in vivo through activating AKT signaling. CD73 is expected to become a new prognostic biomarker and therapeutic target for melanoma.

Objective:To study the expression level of CD73 in cutaneous melanoma and its clinical significance, and to explore its role and molecular mechanism in the occurrence and development of melanoma.Methods:Expression level of the gene NT5E in melanoma was analyzed according to RNA sequencing data from the CCLE database. CD73 expression level was tested in 145 melanoma tissues and corresponding para-tumor tissues by immunohistochemistry tissue microarray. The samples came from 78 male and 67 female patients who received surgical resection and were pathologically diagnosed as melanoma in the department of plastic surgery in Zhongshan Hospital from January of 2008 to December of 2017. The relationship between CD73 expression level and clinicopathological features as well as prognosis was analyzed. Melanoma cell line was transfected with CD73 gene overexpression lentivirus and negative control vectors, respectively. The transfection efficiency was verified by qRT-PCR and Western blot. CCK-8, wound healing and Transwell assays were used to evaluate the proliferation, migration and invasion abilities of the overexpression group and control group. Subcutaneous xenograft tumor models were established in nude mice to assess the tumorigenicity of the two groups. Transcriptome sequencing, GO and KEGG analyses were performed. The expression levels of pAKT and pGSK3β were detected by Western blot. The measurement data were expressed as Mean ±SD. Independent sample t-test was used for comparison between the two groups, and One-way ANOVA was employed for multi-group comparisons. The chi-square test was used for the categorical variables. P<0.05 was considered as statistically significant difference. Results:The expression of CD73 in melanoma was higher than that in para-tumor tissues ( P< 0.05). Higher expression level of CD73 was associated with higher Clark grade ( P= 0.014) and clinical stage ( P= 0.040). The overall survival and disease-free survival of patients with high-CD73-expression were significantly lower than those of patients with low-CD73-expression. CD73 expression, Clark grade and clinical stage are independent risk factors for the poor prognosis of patients with melanoma. Overexpression of CD73 significantly enhanced the proliferation, migration and invasion of melanoma cells in vitro and the tumorigenicity in vivo( P< 0.05). Transcriptome sequencing showed that CD73 was involved in several tumor related signaling pathways, among which the expression of pAKT and pGSK3β was elevated( P< 0.05). Conclusions:CD73 promotes the proliferation, migration and invasion of melanoma cells in vitro and the tumorigenicity in vivo through activating AKT signaling. CD73 is expected to become a new prognostic biomarker and therapeutic target for melanoma.

Objective: To explore the practicability and reproducibility of judgment method and assessment indexes for the end point of double eyelid surgery using 4+ 1 photography in supine position. Methods: From 2017 October to 2018 October, 158 patients were included and randomly divided into 2 groups. Photos were taken by 4 + 1 photography in supine position of 79 patients, while other 79 patients in control group were evaluated by traditional observation. By 4+ 1 photography in supine position, the surgeon stood at the head side of the patient, taking photos with eyes movement: looking straight forward, looking up, looking downward and eye-closed. It was to observe the upper eyelid creases, upper and lower tissues of double eyelid creases, and upper and lower eyelid margo palpebrae. In addition, the surgeon looked from patient′s feet to observe the indexes such as upper margo palpebrae, to make a decision whether the surgery could finish. In control group, the surgeon observed the upper eyelid creases, upper and lower tissues of double eyelid creases and upper and lower eyelid margo palpebrae. The patient has to sit if necessary. Results: All 158 patients were performed double eyelid surgery successfully. Average times of valuating end point was 1.20 by 4+ 1 Photography in supine position, and 1.53 in control group. The operation time of 4+ 1 photography group is (151.65±21.58) s, and control group were (241.53±33.53) s. The satisfaction level was increased to 89.87% in 4+ 1 photography group, compared to 78.48% in control group. Conclusions The 4+ 1 Photography method is simple and easy to practice. The observation indexes are accurate and comprehensive. It is useful in determining the end point of double eyelid plasty in supine position.

Objective To investigate the levels of T helper cell 17 (Th17), Th17-related cytokines in-terleukin 17 (IL-17) and interleukin 23 (IL-23) and regulatory T cell (Treg) in relapsing remitting multiple sclerosis (RRMS). Methods In a case-control study, plasma was collected from RRMS patients (n=20) and healthy subjects as control group (n=20). The percentages of Th17 and Treg cells and the levels of IL-17 and IL-23 were tested. The levels of Th17, Treg, IL-17 and IL-23 of the two groups were compared. Patients were treated with methylprednisolone. The levels of Th17, Treg, IL-17 and IL-23 of multiple sclerosis (MS) patients b efore and after treatment were compared. Expanded disability status scale (EDSS) score and the number of Gd-enhancing lesions were evaluated in the case group. Statistical analysis was made by body mass index (IBM) statistical program for social sciences (SPSS) 17.0 software. Independent sample t test was conducted to compare the measurement data of the case group and the healthy control group, and enumeration data were compared by χ2 test; paired sample t test was performed to compare the data of the case group before and after treatment; Pearson correlation analysis was made forthe variables of the MS group before treatment. Results In the RRMS group, the percentage of Th17 cells in peripheral blood was significantly higher than the control group [(2.10±0.45)％vs (1.09±0.20)％](t=9.130, P<0.01), the levels of Th17-related cytokines IL-17 and IL-23 were remarkably higher than the control group (IL-17:t=19.843, P<0.01;IL-23:t=22.747, P<0.01), and the percentage of Treg cells was significantly lower than the control group [(1.33 ±0.30)％vs (2.52±0.30)％], (t=12.422, P<0.01). The levels of Th17 and IL-17 were positively associated with EDSS score (Th17: r=0.458, P<0.05; IL-17: r=0.480, P<0.05), there was no significant-correlation between the level of IL-23 and EDSS score (r=0.368, P>0.05), and Th17, IL-17 and IL-23 were positively correlated with the number of Gd-enhancing lesions (Th17: r=0.446, P<0.05; IL-17: r=0.544, P<0.05; IL-23: r=0.461, P<0.05). The levels of Th17, IL-17 and IL-23 in the RRMS group after the treatment with methylprednisolone were obviously decreased than before treatment (Th17: t=5.747, P<0.01; IL-17: t=9.967, P<0.01; IL-23: t=14.697, P<0.01), while that of Treg was apparently increased (t=10.050, P<0.01). Compared with the control group, the levels of Th17, IL-17 and IL-23 in the RRMS group after treatment were higher (Th17: t=6.889, P<0.01;IL-17:t=7.185, P<0.01;IL-23:t=13.284, P<0.01), and the percentage of Treg was lower (t=7.622, P<0.01). EDSS score of the RRMS group after treatment was remarkably decreased than before treatment(t=6.190, P<0.01), but the number of Gd-enhanced lesions after treatment was no significantiy changed (t=1.453, P>0.05). Conclusion Th17/Treg expression imbalance and Th17-related cytokines IL-17, IL-23 may participate in the pathological process of MS, and they might be therapeutic target for MS.