Objective : To investigate the brain microvascular tissue block culture method for extracting primary rat brain microvascular endothelial cells and identify its effect. Methods : Brain tissue from 4-week-old Sprague Dawley rats was screened, pre-digested and solidified to obtain brain microvascular segments. These segments were subsequently placed in a CO2incubator for primary culture. The target cells were identified by cell morphology and immunocytochemical staining for factor Ⅷ-related antigen. Results : After a 48-hour culture periodin vitro, the short spindle cells crawled out from around the brain microvascular segments. After 72 hours, island-like cell culsters formed. After 96 hours the clusters fused and the cells formed a typical monolayer, cobble stone-like, and mosaic arrangement. Factor Ⅷ-related antigen immunocytochemical staining showed that the cytoplasm of the cells appeared brown-red, indicating positive expression; DAB stained the nucleus, showing blue-dark. Conclusion The brain microvascular tissue block culture method can isolate and culture primary rat brain microvascular endothelial cells.

Guided by the turbidity toxin theory， it is believed that the key pathogenesis of constipation-predominant irritable bowel syndrome is the obstruction of turbidity toxin and the disruption of intestinal function. Treatment is based on the principles of dispelling turbidity toxin and promoting intestinal function. The clinical patterns can be divided into three types， turbidity toxin heat accumulation pattern， turbidity toxin combined with liver depression and qi stagnation pattern， and turbidity toxin combined with qi and yin deficiency pattern. The treatment can respectively use self-prescribed Tongfu Jiangzhuo Formula （通腑降浊方） to clear heat and unblock the bowels， direct the turbid downward and resolve toxins； use self-prescribed Shugan Jiangzhuo Formula （疏肝降浊方） to soothe the liver and move qi， direct the turbid downward and resolve toxins； use self-prescribed Mazhi Jiangzhuo Formula （麻枳降浊方） to boost qi and nourish yin， moisten the intestines to remove turbidity and resolve toxins.

The Chinese hamster ovary (CHO) cell is the most representative mammalian cell protein expression system, and it is widely used in recombinant protein, vaccine and other biopharmaceutical fields. However, due to its vulnerability to environmental factors, apoptosis, and metabolic inhibitors, CHO cells demonstrate poor robustness, and thus the integrated viable cell density and unit cell productivity are largely limited. To improve the robustness and foreign protein expression efficiency of CHO cells, we employed CRISPR/Cas9 to knock out the apoptosis genes Bax and Bak and the lactate dehydrogenase gene LDHa, thereby blocking apoptosis and lactic acid metabolism pathways. The results of apoptosis and single cell viability detection showed that the number of apoptotic cells in the knockout cell lines Bax^-/-, Bax-bak^-/-, and LDHa-Bax-bak^-/- was reduced by 22.51%, 37.73%, and 64.12%, respectively, compared with the wild-type cell line CHO-K1, which indicated that the anti-apoptotic ability was significantly improved. After staurosporine treatment, the single cell viability of Bax^-/-, Bax-bak^-/-, and LDHa-Bax-bak^-/- cells was increased by 30.8%, 22%, and 41.1%, respectively. After treatment with puromycin, the single cell viability of Bax^-/-, Bax-bak^-/-, and LDHa-Bax-bak^-/- cells was increased by 26.7%, 30.7%, and 38.8%, respectively. To further investigate the production performance of cells obtained after blocking apoptosis and lactic acid metabolism pathways, we induced transient expression of human tissue plasminogen activator (tPA) in these cells. The results showed that the secretion of tPA in Bax^-/-, Bax-Bak^-/-, and LDHa-Bax-Bak^-/- cells was 11.12%, 46.18%, and 63.13%, respectively, higher than that in wild-type CHO-K1 cells. The expression of intracellular tPA was increased by 35.65%, 130%, and 192.15%. In conclusion, blocking apoptosis and lactic acid metabolism pathways simultaneously can improve cell robustness and productivity, with the performance better than blocking the apoptosis pathway alone. The above results indicated that the constructed cell lines were expected to be the delivery carriers of protein drugs such as medicinal peptides, and better used for the treatment of diseases.
CHO Cells ; Cricetulus ; Animals ; Apoptosis/genetics* ; Lactic Acid/metabolism* ; Recombinant Proteins/biosynthesis* ; L-Lactate Dehydrogenase/genetics* ; bcl-2-Associated X Protein/genetics* ; bcl-2 Homologous Antagonist-Killer Protein/genetics* ; Cricetinae ; CRISPR-Cas Systems ; Staurosporine/pharmacology*

BACKGROUND: The purpose of this study was to evaluate the safety and efficacy of subsequent radiotherapy (RT) following first-line treatment with durvalumab plus chemotherapy in patients with extensive-stage small cell lung cancer (ES-SCLC). METHODS: A total of 122 patients with ES-SCLC from three hospitals during July 2019 to December 2021 were retrospectively analyzed. Inverse probability of treatment weighting (IPTW) analysis was performed to address potential confounding factors. The primary focus of our evaluation was to assess the impact of RT on progression-free survival (PFS) and overall survival (OS). RESULTS: After IPTW analysis, 49 patients received durvalumab plus platinum-etoposide (EP) chemotherapy followed by RT (Durva + EP + RT) and 72 patients received immunochemotherapy (Durva + EP). The median OS was 17.2 months vs . 12.3 months (hazard ratio [HR]: 0.38, 95% confidence interval [CI]: 0.17-0.85, P = 0.020), and the median PFS was 8.9 months vs . 5.9 months (HR: 0.56, 95% CI: 0.32-0.97, P = 0.030) in Durva + EP + RT and Durva + EP groups, respectively. Thoracic radiation therapy (TRT) resulted in longer OS (17.2 months vs . 14.7 months) and PFS (9.1 months vs . 7.2 months) compared to RT directed to other metastatic sites. Among patients with oligo-metastasis, RT also showed significant benefits, with a median OS of 17.4 months vs . 13.7 months and median PFS of 9.8 months vs . 5.9 months compared to no RT. Continuous durvalumab treatment beyond progression (TBP) prolonged OS compared to patients without TBP, in both the Durva + EP + RT (NA vs . 15.8 months, HR: 0.48, 95% CI: 0.14-1.63, P = 0.238) and Durva + EP groups (12.3 months vs . 4.3 months, HR: 0.29, 95% CI: 0.10-0.81, P = 0.018). Grade 3 or 4 adverse events occurred in 13 (26.5%) and 13 (18.1%) patients, respectively, in the two groups; pneumonitis was mostly low-grade. CONCLUSION Addition of RT after first-line immunochemotherapy significantly improved survival outcomes with manageable toxicity in ES-SCLC.
Humans ; Small Cell Lung Carcinoma/therapy* ; Retrospective Studies ; Male ; Female ; Middle Aged ; Lung Neoplasms/therapy* ; Aged ; Antibodies, Monoclonal/therapeutic use* ; Adult ; Immunotherapy/methods* ; Aged, 80 and over

Many female patients with inflammatory bowel disease (IBD) are of reproductive age, and addressing fertility-related issues is an important part of comprehensive treatment. Although the fertility of female IBD patients does not differ significantly from that of the general population, many women with IBD opt for childlessness. This voluntary childlessness may stem from concerns about the adverse effects of IBD and its treatment on fertility, pregnancy, and newborns. A lack of in depth understanding of fertility, socioeconomic status, demographic factors, and insufficient professional medical advice may also contribute to patients' decision to choose voluntary childlessness. This article analyzes the factors influencing voluntary childlessness in female IBD patients and summarizes recommendations aimed at reducing trend, to enhance fertility awareness among female IBD patients and providing useful references for developing reasonable fertility plans.

Objectives:To establish an efficient in vitro culture method for spinal cord microvascular en-dothelial cells(SCMECs)of rabbits.Methods:15 Japanese large-ear white rabbits(2-week-old,either sex)were selected.Under sterile conditions,the spinal cord was obtained by incising the back skin,muscle layers and vertebral canal along its longitudinal axis.The spinal cord was minced,and myelin impurities were removed by density gradient centrifugation using bovine serum albumin.The spinal cord microvascular segments were obtained by filtration through a sieve.The enzymatic digestion method was used,with 0.1％type Ⅱ collage-nase digesting the microvascular segments for 20min,after which,the segments were seeded into culture flasks,and then placed in a CO2 incubator for static culture for 72h.The above procedures were performed with five rabbits at a time,repeated three times,and the target cells from the third culture were taken as the objects of observation and identification.The growth and morphological characteristics of the cells were ob-served under an inverted phase-contrast microscope.Immunocytochemical staining was used to detect the ex-pression of factor Ⅷ-related antigen to identify the cultured SCMECs.Results:Microscopically,the segments showed short-rod or branched shapes post-seeding.After 24h of culture,short spindle-shaped cells migrated out from around the microvascular segments and grew in a"budding"manner.After 48h,"island-like"cell colonies formed.After 72h,the cells covered the bottom of the dish,growing in a typical monolayer,"paving stone-like"mosaic pattern.Immunocytochemical staining showed that over 99％of the cells had brownish-red cytoplasm,with positive expression of factor Ⅷ-related antigen,hematoxylin stained the nuclei and was shown in blue.Conclusions:Enzymatic digestion can successfully isolate and culture SCMECs of Japanese large-ear white rabbits with good activity and high purity.

Objective:To establish a simple and efficient method for in vitro extraction,culture and identification of brain microvascular endothelial cells(BMECs)of rabbits.Method:The cerebral cortex of 3-week-old rabbits was cut up,sieved and digested with type II collagenase to obtain pure brain microvascular segments for primary culture and identification.Results:The cultured target cells grew in a typical"cobblestone-like"arrangement.Immunohistochemi-cal staining for von Willebrand factor(vWF)related antigen showed that the cytoplasm was presented as brownish red and the expression was positive.Conclusion:The physical sieving method combined with the chemical enzyme diges-tion method can simply and efficiently culture primary rabbit BMECs with strong activity and high purity.

Objectives:To establish an efficient in vitro culture method for spinal cord microvascular en-dothelial cells(SCMECs)of rabbits.Methods:15 Japanese large-ear white rabbits(2-week-old,either sex)were selected.Under sterile conditions,the spinal cord was obtained by incising the back skin,muscle layers and vertebral canal along its longitudinal axis.The spinal cord was minced,and myelin impurities were removed by density gradient centrifugation using bovine serum albumin.The spinal cord microvascular segments were obtained by filtration through a sieve.The enzymatic digestion method was used,with 0.1％type Ⅱ collage-nase digesting the microvascular segments for 20min,after which,the segments were seeded into culture flasks,and then placed in a CO2 incubator for static culture for 72h.The above procedures were performed with five rabbits at a time,repeated three times,and the target cells from the third culture were taken as the objects of observation and identification.The growth and morphological characteristics of the cells were ob-served under an inverted phase-contrast microscope.Immunocytochemical staining was used to detect the ex-pression of factor Ⅷ-related antigen to identify the cultured SCMECs.Results:Microscopically,the segments showed short-rod or branched shapes post-seeding.After 24h of culture,short spindle-shaped cells migrated out from around the microvascular segments and grew in a"budding"manner.After 48h,"island-like"cell colonies formed.After 72h,the cells covered the bottom of the dish,growing in a typical monolayer,"paving stone-like"mosaic pattern.Immunocytochemical staining showed that over 99％of the cells had brownish-red cytoplasm,with positive expression of factor Ⅷ-related antigen,hematoxylin stained the nuclei and was shown in blue.Conclusions:Enzymatic digestion can successfully isolate and culture SCMECs of Japanese large-ear white rabbits with good activity and high purity.

Objective:To establish a simple and efficient method for in vitro extraction,culture and identification of brain microvascular endothelial cells(BMECs)of rabbits.Method:The cerebral cortex of 3-week-old rabbits was cut up,sieved and digested with type II collagenase to obtain pure brain microvascular segments for primary culture and identification.Results:The cultured target cells grew in a typical"cobblestone-like"arrangement.Immunohistochemi-cal staining for von Willebrand factor(vWF)related antigen showed that the cytoplasm was presented as brownish red and the expression was positive.Conclusion:The physical sieving method combined with the chemical enzyme diges-tion method can simply and efficiently culture primary rabbit BMECs with strong activity and high purity.

Many female patients with inflammatory bowel disease (IBD) are of reproductive age, and addressing fertility-related issues is an important part of comprehensive treatment. Although the fertility of female IBD patients does not differ significantly from that of the general population, many women with IBD opt for childlessness. This voluntary childlessness may stem from concerns about the adverse effects of IBD and its treatment on fertility, pregnancy, and newborns. A lack of in depth understanding of fertility, socioeconomic status, demographic factors, and insufficient professional medical advice may also contribute to patients' decision to choose voluntary childlessness. This article analyzes the factors influencing voluntary childlessness in female IBD patients and summarizes recommendations aimed at reducing trend, to enhance fertility awareness among female IBD patients and providing useful references for developing reasonable fertility plans.