[摘 要] 目的：探讨RNA结合基序蛋白15（RBM15）是否通过含跨膜Bax抑制基序蛋白6（TMBIM6）/TGF-β/Smad轴促进宫颈癌细胞增殖、迁移及侵袭。方法：收集2023年1月至2025年1月在苏州大学附属张家港医院接受手术治疗的82例宫颈癌患者的肿瘤组织与癌旁组织。应用R语言分析癌症基因组图谱（TCGA）数据库中RBM15、TMBIM6 mRNA的表达；采用免疫组织化学法和Western blotting法检测RBM15、TMBIM6蛋白表达；采用Kaplan-Meier曲线分析RBM15、TMBIM6 mRNA表达与宫颈癌患者预后的关系；采用CCK-8法、划痕实验及Transwell侵袭实验检测RBM15、TMBIM6对宫颈癌细胞增殖、迁移及侵袭的影响；利用基于序列的RNA腺苷甲基化位点预测工具（SRAMP）预测TMBIM6 mRNA的N6-甲基腺苷（m6A）修饰位点；采用RNA免疫共沉淀（RIP）实验、RNA衰变和挽救实验鉴定RBM15与TMBIM6 mRNA的相互作用；采用甲基化RNA免疫共沉淀定量PCR（MeRIP-qPCR）检测TMBIM6 mRNA的m6A富集；采用Western blotting法检测RBM15通过TMBIM6对宫颈癌细胞TGF-β/Smad通路相关蛋白表达的影响。结果：宫颈癌组织中RBM15 mRNA、TMBIM6 mRNA表达均高于癌旁组织（均P < 0.05），癌组织RBM15、TMBIM6阳性率均高于癌旁组织（均P < 0.001）。宫颈癌细胞系HeLa、MS-751、SiHa中RBM15、TMBIM6蛋白表达均高于正常宫颈上皮细胞系Ect1/E6E7（均P < 0.05）。国际妇产科联盟（FIGO）分期ⅠB2～ⅡB期宫颈癌组织中RBM15、TMBIM6蛋白阳性率高于ⅠA～ⅠB1期（P均 < 0.05）。RBM15 mRNA高表达组、TMBIM6 mRNA高表达组5年无进展生存率分别低于各自的低表达组（均P < 0.05）。宫颈癌组织中RBM15 mRNA与TMBIM6 mRNA表达呈正相关（P < 0.05）。SRAMP预测结果显示，TMBIM6 mRNA序列的960、24876、25203位点为高可信度m6A修饰位点。在HeLa、SiHa细胞中过表达RBM15后，TMBIM6 mRNA和蛋白表达升高（P < 0.05）；敲低RBM15后，TMBIM6 mRNA和蛋白表达降低（P < 0.05）。与IgG组相比，RBM15抗体免疫沉淀物中TMBIM6 mRNA显著富集。MeRIP-qPCR结果显示，TMBIM6 mRNA的960、24876、25203位点均存在明显的m6A富集。敲低RBM15可缩短TMBIM6 mRNA半衰期并降低其稳定性。敲低RBM15可抑制宫颈癌细胞增殖、迁移及侵袭，过表达TMBIM6可逆转上述影响。在HeLa、SiHa细胞中过表达TMBIM6后，TGF-β1、Smad2、Smad3蛋白表达升高；敲低RBM15后，上述蛋白表达降低，过表达TMBIM6可逆转该影响。结论：RBM15可能通过m6A修饰提高TMBIM6 mRNA稳定性并激活TGF-β/Smad通路，从而促进宫颈癌细胞增殖、迁移及侵袭。

Objective: To establish a method for determining the molecular weight and molecular weight distribution of Poly(Lactide-co-Glycolide Acid) (PLGA) using Size Exclusion Chromatography-Refractive Index-Multiangle Laser Light Scattering (SEC-RI-MALLS) and Size Exclusion Chromatography-Refractive Index (SEC-RID), and to compare the results obtained from these two methods.
Methods: For SEC-RI-MALLS, tetrahydrofuran was used as the mobile phase, Shodex GPC KF-803L was employed as the chromatographic column with a flow rate of 1 mL·min^-1, column temperature at 30 ℃, and an injection volume of 100 μL. For SEC-RID, tetrahydrofuran was also used as the mobile phase, Agilent PLgel 5 μm MIXD-D was used as the chromatographic column with a flow rate of 1 mL·min^-1, column temperature at 30 ℃, differential detector temperature at 35 ℃, and an injection volume of 20 μL. The molecular weight and molecular weight distribution were calculated using Agilent’s GPC software. The newly established methods were validated methodologically, and the molecular weight and molecular weight distribution of 13 batches of samples were determined.
Results: The precision, accuracy, stability, and repeatability tests for SEC-RI-MALLS showed RSD values of 1.35%, 1.58%, 1.53%, and 1.26%, respectively. The SEC-RID method exhibited good linearity (r=0.999 9), with RSD values for precision, accuracy, stability, and repeatability tests (n=6) of 2.05%, 1.62%, 1.30%, and 2.97%, respectively. The results obtained from SEC-RI-MALLS were lower than those from SEC-RID, and the molecular weight distribution coefficient was smaller, but the results from the paired T-test performed with the value measured by SEC-RID method and the value measured by SEC-RI-MALLS method multiplied a conversion coefficient of 1.5 showed no significant difference between the two methods.
Conclusion: Both methods are stable and reliable, and can be used for the determination of PLGA molecular weight and molecular weight distribution based on the specific situations.

Objective:To investigate the effect of deubiquitinating enzymes(DUBs) USP2 on depressive-like behavior and hippocampal NF-κB expression in mice.Methods:(1) USP2 silencing experiment: Two USP2 silencing interference sequences with the highest knockdown efficiency were screened and cloned into a lentivirus vector. Mice were microinjected with lentivirus vector into both sides of hippocampus to silence the USP2 gene, and depressive behavior and USP2 protein expression in hippocampal tissue were observed. (2) Venlafaxine intervention experiment: total 32 healthy male C57BL/6 mice were randomly divided into virus control group, Venlafaxine group, USP2 silencing group, and USP2 silencing+ Venlafaxine group according to the random number table method, with 8 mice in each group. Mice were injected with lentivirus into both side of the hippocampus, and 7 days later, they were given intraperitoneal injection of Venlafaxine (5 mg/kg, once a day, for a total of 14 days). After the administration, the depressive behavior of mice was detected by forced swimming test(FST) and tail suspension test(TST), and the expression levels of USP2, p-IκBα, IκBα, p-NF-κB p65, and NF-κB p65 in the hippocampus of mice were detected by Western blot.SPSS 25.0 and GraphPad Prism 8.0 were used for data processing and chart drawing.The t-test was used for comparison between two groups, One-way ANOVA was used for comparison among multiple groups, and Tukey HSD or LSD- t was used for post hoc pairwise comparison when there was homogeneity of variance. Results:(1)The results of the USP2 silencing experiment showed that both screened USP2 silencing sequences had good gene knockout effects. The expression levels of USP2 protein in the hippocampus of mice injected with USP2 silencing virus were lower than those of the negative control virus groups (both P<0.05). The immobility time of mice in the FST and TST was higher than that of the negative control virus group (both P<0.05). (2)Venlafaxine intervention experiment: There were statistically significant differences in immobility time among the four groups of mice in the FST and TST ( F=8.90, 4.41, both P<0.05). The immobility time of FST and the immobility time of TST in the USP2 silencing+ Venlafaxine group ((48.13±12.76) s, (77.38±12.35) s) were lower than those in the USP2 silencing group((129.88±11.67)s, (148.29±15.31)s) (both P<0.05). There were statistically significant differences in the expression levels of USP2, p-IκBα, and p-NF-κB p65 proteins in the hippocampal tissues of the four groups of mice ( F=8.39, 5.78, 21.32, all P<0.05).The expression level of USP2 protein in the USP2 silencing group(0.49±0.07) was lower than that in the USP2 silencing+ Venlafaxine group(0.79±0.08) and virus control group(1.00±0.07)(both P<0.05), while the expression levels of p-IκBα, p-NF-κB p65 protein (1.63±0.18, 2.14±0.24) were higher than those in the virus control group (1.00±0.06, 1.00±0.04) and the USP2 silencing+ Venlafaxine group (0.70±0.23, 0.68±0.09) (both P<0.05). Conclusion:USP2 scilencing can induce depressive-like behaviors in mice. Venlafaxine ameliorates USP2 silencing-induced depressive-like behaviors, which may be associated with the hippocampal NF-κB signaling pathway.

Objective To systematically review the risk factors for liver injury in breast cancer patients after chemotherapy.Methods PubMed,Cochrane Library,Embase,Web of Science,CNKI,SinoMed,WanFang Data and VIP databases were electronically searched to collect studies on the influential factors of liver injury in breast cancer patients from inception to March 31,2024.Two reviewers independently screened the literature,extracted data and evaluated the risk of bias of the included studies.Meta-analysis was performed by using RevMan 5.3 software.Results A total of 15 studies involving 6,486 patients were included.The results of Meta-analysis showed that hepatitis B virus infection[OR=2.41,95％CI(1.52,3.82),P＜0.001],lymph node metastasis[OR=1.27,95％CI(1.05,1.55),P=0.02],hyperlipidemia[OR=1.58,95％CI(1.13,2.20)P=0.007],liver metastasis[OR=3.19,95％CI(2.11,4.84),P＜0.001],tumor node metastasis(TNM)stage Ⅲ to Ⅳ[OR=1.49,95％CI(1.17,1.90),P＜0.001],were risk factors for liver injury after breast cancer chemotherapy.Compared with other chemotherapy agents/regimens,the use of fluorouracil[OR=0.58,95％CI(0.42,0.80),P＜0.001],cyclophosphamide[OR=0.65,95％CI(0.47,0.89),P=0.008],AC regimen[OR=0.38,95％CI(0.22,0.66),P＜0.00 1]and neoadjuvant chemotherapy[OR=0.61,95％CI(0.43,0.88),P=0.007]were associated with a lower risk of liver injury,the use of paclitaxel drugs[OR=2.85,95％CI(2.31,3.52),P＜0.001],intensive regimen[OR=3.48,95％CI(2.15,5.62),P＜0.001],TAC regimen[OR=2.42,95％CI(1.38,4.24),P=0.002]increased the risk of liver injury.Tumor TNM stage I[OR=0.63,95％CI(0.51,0.78),P=0.001]were protective factors for liver injury after chemotherapy in breast cancer patients.Conclusion The current evidence shows that multiple factors influence the incidence of liver injury after breast cancer chemotherapy.Medical staff can formulate targeted chemotherapy programs according to the influencing factors and clinical characteristics to reduce the risk of liver injury after chemotherapy.

Objective To investigate the changes and diagnostic value of serum hypoxia inducible factor 1 subunit alpha(HIF-1α)and Toll-like receptor 4(TLR4)levels in patients with chronic obstructive pulmonary disease(COPD)complicated with pulmonary Aspergillosis infection.Methods A total of 240 COPD patients who visited Xi'an Qinhuang Hospital(hereinafter referred to as the hospital)from December 2020 to Decem-ber 2023 were selected as the study subjects in the study,and another 218 volunteers who underwent physical examinations at the hospital were selected as the control group.The COPD patients were separated into an in-fected group(124 cases)and an uninfected group(116 cases)based on whether they had pulmonary Aspergil-losis infection.Enzyme-linked immunosorbent assay was applied to detect the levels of HIF-1α and TLR4 in patients.Fully automated biochemical analyzer was applied to detect lactate dehydrogenase(LDH)and albu-min(ALB)levels.Multivariate Logistic regression was applied to analyze the influencing factors of infection in COPD patients.Pearson correlation was applied to analyze the correlation between HIF-1α and TLR4 levels in the infected group.Receiver operating characteristic(ROC)curve was applied to analyze the diagnostic val-ue of HIF-1α and TLR4 levels for the occurrence of infection in COPD patients.Results Compared with the control group,the COPD group showed an increase in HIF-1α and TLR4 levels(P＜0.05).Compared with the uninfected group,the proportion of dyspnea,antibiotics＞3 types,the duration of antibiotic use ≥ 14 days,mechanical ventilation procedures,the longer glucocorticosteroid(GC)use time,and levels of LDH,HIF-1α,TLR4 in the infected group were higher(P＜0.05),while the level of ALB was lower(P＜0.05).The types of antibiotics＞3 types,the duration of antibiotic use ≥ 14 days,the duration of GC use,and elevat-ed levels of LDH,HIF-1α,and TLR4 were independent risk factors for infection in COPD patients(P＜0.05),while elevated level of ALB was an independent protective factor for infection in COPD patients(P＜0.05).The levels of HIF-1α and TLR4 in the infected group were positively correlated(r=0.453,P＜0.001).The area under the curve(AUC)of HIF-1α and TLR4 in diagnosing infection in COPD patients alone was 0.816 and 0.813,and the AUC of their combined diagnosis was 0.930,which was better than their indi-vidual diagnoses(Zcombination-HIF-1α=4.923,Z combination-TLR4=5.192,P＜0.001,P＜0.001).Conclusion The levels of HIF-1α and TLR4 increase in COPD patients,and further increase after infection with pulmonary Aspergil-lus.They are independent risk factors for infection in patients,and the two are positively correlated.The combined di-agnosis of pulmonary aspergillosis has certain value and provides a theoretical basis for clinical diagnosis.

Objective To systematically review the risk factors for liver injury in breast cancer patients after chemotherapy.Methods PubMed,Cochrane Library,Embase,Web of Science,CNKI,SinoMed,WanFang Data and VIP databases were electronically searched to collect studies on the influential factors of liver injury in breast cancer patients from inception to March 31,2024.Two reviewers independently screened the literature,extracted data and evaluated the risk of bias of the included studies.Meta-analysis was performed by using RevMan 5.3 software.Results A total of 15 studies involving 6,486 patients were included.The results of Meta-analysis showed that hepatitis B virus infection[OR=2.41,95％CI(1.52,3.82),P＜0.001],lymph node metastasis[OR=1.27,95％CI(1.05,1.55),P=0.02],hyperlipidemia[OR=1.58,95％CI(1.13,2.20)P=0.007],liver metastasis[OR=3.19,95％CI(2.11,4.84),P＜0.001],tumor node metastasis(TNM)stage Ⅲ to Ⅳ[OR=1.49,95％CI(1.17,1.90),P＜0.001],were risk factors for liver injury after breast cancer chemotherapy.Compared with other chemotherapy agents/regimens,the use of fluorouracil[OR=0.58,95％CI(0.42,0.80),P＜0.001],cyclophosphamide[OR=0.65,95％CI(0.47,0.89),P=0.008],AC regimen[OR=0.38,95％CI(0.22,0.66),P＜0.00 1]and neoadjuvant chemotherapy[OR=0.61,95％CI(0.43,0.88),P=0.007]were associated with a lower risk of liver injury,the use of paclitaxel drugs[OR=2.85,95％CI(2.31,3.52),P＜0.001],intensive regimen[OR=3.48,95％CI(2.15,5.62),P＜0.001],TAC regimen[OR=2.42,95％CI(1.38,4.24),P=0.002]increased the risk of liver injury.Tumor TNM stage I[OR=0.63,95％CI(0.51,0.78),P=0.001]were protective factors for liver injury after chemotherapy in breast cancer patients.Conclusion The current evidence shows that multiple factors influence the incidence of liver injury after breast cancer chemotherapy.Medical staff can formulate targeted chemotherapy programs according to the influencing factors and clinical characteristics to reduce the risk of liver injury after chemotherapy.

Objective:To investigate the effect of deubiquitinating enzymes(DUBs) USP2 on depressive-like behavior and hippocampal NF-κB expression in mice.Methods:(1) USP2 silencing experiment: Two USP2 silencing interference sequences with the highest knockdown efficiency were screened and cloned into a lentivirus vector. Mice were microinjected with lentivirus vector into both sides of hippocampus to silence the USP2 gene, and depressive behavior and USP2 protein expression in hippocampal tissue were observed. (2) Venlafaxine intervention experiment: total 32 healthy male C57BL/6 mice were randomly divided into virus control group, Venlafaxine group, USP2 silencing group, and USP2 silencing+ Venlafaxine group according to the random number table method, with 8 mice in each group. Mice were injected with lentivirus into both side of the hippocampus, and 7 days later, they were given intraperitoneal injection of Venlafaxine (5 mg/kg, once a day, for a total of 14 days). After the administration, the depressive behavior of mice was detected by forced swimming test(FST) and tail suspension test(TST), and the expression levels of USP2, p-IκBα, IκBα, p-NF-κB p65, and NF-κB p65 in the hippocampus of mice were detected by Western blot.SPSS 25.0 and GraphPad Prism 8.0 were used for data processing and chart drawing.The t-test was used for comparison between two groups, One-way ANOVA was used for comparison among multiple groups, and Tukey HSD or LSD- t was used for post hoc pairwise comparison when there was homogeneity of variance. Results:(1)The results of the USP2 silencing experiment showed that both screened USP2 silencing sequences had good gene knockout effects. The expression levels of USP2 protein in the hippocampus of mice injected with USP2 silencing virus were lower than those of the negative control virus groups (both P<0.05). The immobility time of mice in the FST and TST was higher than that of the negative control virus group (both P<0.05). (2)Venlafaxine intervention experiment: There were statistically significant differences in immobility time among the four groups of mice in the FST and TST ( F=8.90, 4.41, both P<0.05). The immobility time of FST and the immobility time of TST in the USP2 silencing+ Venlafaxine group ((48.13±12.76) s, (77.38±12.35) s) were lower than those in the USP2 silencing group((129.88±11.67)s, (148.29±15.31)s) (both P<0.05). There were statistically significant differences in the expression levels of USP2, p-IκBα, and p-NF-κB p65 proteins in the hippocampal tissues of the four groups of mice ( F=8.39, 5.78, 21.32, all P<0.05).The expression level of USP2 protein in the USP2 silencing group(0.49±0.07) was lower than that in the USP2 silencing+ Venlafaxine group(0.79±0.08) and virus control group(1.00±0.07)(both P<0.05), while the expression levels of p-IκBα, p-NF-κB p65 protein (1.63±0.18, 2.14±0.24) were higher than those in the virus control group (1.00±0.06, 1.00±0.04) and the USP2 silencing+ Venlafaxine group (0.70±0.23, 0.68±0.09) (both P<0.05). Conclusion:USP2 scilencing can induce depressive-like behaviors in mice. Venlafaxine ameliorates USP2 silencing-induced depressive-like behaviors, which may be associated with the hippocampal NF-κB signaling pathway.

OBJECTIVE To establish HPLC methods with different separation principles to analyze the relevant impurities in the APIs of bivalirudin from seven enterprises, to provide a basis for the comprehensive control of related substances of bivalirudin. METHODS Reversed-phase high performance liquid chromatography(RP-HPLC) was used to separate and analyze 11 kinds of impurities. Hydrophilic chromatography(HILIC)-HPLC was used to control four process impurities. Polymers were determined by size exclusion chromatography(SEC)-HPLC. RESULTS The established RP-HPLC could effectively separate the principal component and 11 impurities, the correction factors of 11 impurities were between 0.8−1.2, the detection concentration of bivalirudin was 0.1 μg·mL−1, and the detection limit was 0.004%. The established HILIC-HPLC could effectively separate the principal components and four process impurities, and the detection concentration of bivalirudin was 0.3 μg·mL−1, and the detection limit was 0.01%. Under SEC-HPLC conditions, the polymer and bivalirudin peaked sequentially, the resolution of the two was 2.9, the detection concentration of bivalirudin was 6 ng·mL−1, and the detection limit was 0.000 6%. Fifteen kinds of known impurities and polymers in 15 batches of samples from 7 enterprises were calculated by the self-control method of principal components, and the impurity contents from different enterprises had a certain correlation with their production processes. CONCLUSION The three different principles of the method have good specificity, high sensitivity, good durability, and reliable results, and can be used for quality control of substances related to bivalirudin.

Objective:To established a rapid method for the determination of lactose(C12 H22 O11)content.Methods:The specific rotation([α]20D)of pure lactose was measured with lactose extract product,and the correc-tion coefficient was calculated.The measured rotatory value of the sample was multiplied by the correction coeffi-cient to obtain the mass(g)of lactose in the test product,and the lactose content was calculated.The equivalence of the newly established polarimetric method and the legal test method(HPLC-RID method)for the determination of lactose content was studied.Results:The linear relation of established method was excellent with the range of 0.05-0.20 g·mL-1(r=1.000 0)and the method also had good reproducibility(RSD=0.07％(n=6)).The lactose content of 141 batches samples measured by polarimetry was compared with the results determined by official analytical procedure,and the results showed the relative deviation between the same batches was less than 1.0％.The results of one-way ANOVA also showed that there was no significant difference between two groups(Sig.＞0.05).Conclusion:The performance of polarimetry is comparable to HPLC-RID in the Chinese Pharma-copoeia 2020 Vol Ⅳ.Meanwhile,the polarimetry can reduce the test cost,shorten the test time and meet the requirements of the quality control.

Intervertebral disc degeneration is the most common cause of chronic low back pain and the leading cause of disability in adults. The fact that lacking of effective treatment methods often causes a serious economic and social burden. Intervertebral disc degeneration is the result of multifactorial factors. The prevalence of intervertebral disc degeneration increases drastically with age, what is more, mechanical trauma, genetic predisposition,lifestyle factors and certain metabolic disorders. At present, the main treatment methods both pharmacological and surgical interventions just aim at relieving symptoms and improving function, and can not fundamentally reverse the process of intervertebral disc degeneration, which not only bring inevitable side effects and high cost, but also the long-term curative effect is limited. In theory, biological therapy can not only reverse or delay the process of it, but also can maximize preservation and restore the normal physiological function of the disc, which has been the focus and hot spot areas of research in recent years. The methods of inhibiting inflammation, promote the proliferation and division of residual cells, stem cell transplantation, cell scaffolds and new biomaterials all provide new ideas and direction for the treatment of intervertebral disc degeneration. This paper makes a review of the research progress in related fields, in order to provide a valuable reference for the selection of intervertebral disc degeneration treatment options.