Objective:To explore the clinicopathological characteristics and prognosis of fumarate hydratase-deficient uterine leiomyoma (FH-dUL).Methods:Clinical data and follow-up information for 117 patients with FH-dUL diagnosed through surgical pathology and immunohistochemistry in the First Affiliated Hospital of Nanjing Medical University from January 2020 to December 2024, were collected. A control group of 130 patients with common uterine leiomyomas was also included. The differences between the two groups in clinical, imaging, and pathological characteristics were compared. Additionally, recurrence rates, fertility outcomes for FH-dUL patients, and the incidence of renal cancer in FH germline mutation carriers were monitored.Results:(1) Comparison of clinicopathological characteristics: the median age of 117 FH-dUL patients was 35 years, and the median age at first diagnosis of uterine leiomyomas was 29 years, both significantly younger than the control group (41 and 36 years; both P<0.01). The FH-dUL group showed significantly higher incidences of uterine myomectomy, multiple leiomyomas, diffusion restriction on pelvic magnetic resonance imaging diffusion weighted imaging, and typical pathological features (candelabra-like vessels, bizarre nuclei, cytoplasmic eosinophilic globules, perinuclear halo, cellular atypia) and higher ultrasound blood flow score (all P<0.05). Of the 30 FH-dUL patients who underwent genetic testing, 9 had germline mutations, 3 had somatic mutations, and 6 had mutations of unclear origin. Among the 9 FH gene germline mutation patients, 2 had already developed renal cell carcinoma. (2) Recurrence analysis: among the 56 patients who underwent uterine myomectomy, 22 (39.3%, 22/56) experienced recurrence during follow-up, compared to 12 (21.8%, 12/55) of the 55 patients in the control group, the difference between the two groups was statistically significant ( P=0.046). Multivariate binary logistic regression analysis showed that cellular leiomyomas ( OR=9.489, 95% CI: 1.740-51.755; P=0.009) and multiple uterine leiomyomas ( OR=10.709, 95% CI: 1.354-84.683; P=0.025) were significant risk factors for recurrence in FH-dUL. (3) Fertility analysis: among the 66 FH-dUL patients who underwent fertility-preserving surgery, 16 had the intention to have fertility desire, only 2 (2/16) completed their fertility plans during follow-up. Conclusions:Clinicopathological features and imaging features help to differentiate FH-dUL from common type uterine fibroids, but lack specificity, and the diagnosis of FH-dUL is based on immunohistochemistry. The recurrence rate after resection of FH-dUL is high, and cellular and multiple leiomyomas are important predictors of recurrence. It is crucial to perform genetic testing, genetic counseling, drug treatment to prevent recurrence, fertility guidance, and long-term comprehensive management after surgery for FH-dUL management.

This study aims to explore the mechanism of Huanglian Jiedu Decoction(HJD) in treating acute liver injury(ALI) in the mouse model of sepsis induced by lipopolysaccharide(LPS). Fifty-four male C57BL/6 mice were randomized into six groups: blank group, model group, low-, medium-, and high-dose group HJD, and dexamethasone group. The mouse model of sepsis was established by intraperitoneal injection of LPS after 7 days of gavage with HJD, and dexamethasone(0.2 mL) was injected intraperitoneally 1.5 h after modeling. The murine sepsis score(MSS) was recorded 12 h after modeling. The levels of alanine aminotransferase(ALT) and aspartate aminotransferase(AST) in the liver tissue and tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6) in the serum were measured by ELISA. Hematoxylin-eosin(HE) staining was used to observe the pathological changes of the mouse liver. The content of light chain 3 of microtubule-associated protein 1(LC3) was detected by immunofluorescence, and that of sirtuin 1(SIRT1) was detected by immunohistochemistry. The mRNA levels of adenosine 5'-monophosphate-activated protein kinase(AMPK), LC3, and P62 were detected by RT-PCR. Western blot was employed to determine the protein levels of AMPK, p-AMPK, and SIRT1 in the liver tissue. The results showed that compared with model group, drug interventions decreased the MSS and liver injury indicators, lowered the levels of inflammatory cytokines, improved the liver tissue structure, upregulated the protein levels of of p-AMPK/AMPK and SIRT1 and the mRNA levels of AMPK and LC3, and downregulated the mRNA level of P62. To sum up, HJD can regulate the autophagy level and reduce inflammation to ameliorate acute liver injury in septic mice by activating the AMPK/SIRT1 autophagy pathway.
Animals ; Drugs, Chinese Herbal/administration & dosage* ; Sirtuin 1/genetics* ; Male ; Mice ; Sepsis/metabolism* ; Mice, Inbred C57BL ; Autophagy/drug effects* ; AMP-Activated Protein Kinases/genetics* ; Liver/metabolism* ; Humans ; Signal Transduction/drug effects* ; Disease Models, Animal ; Tumor Necrosis Factor-alpha/genetics*

Objective:To explore the clinicopathological characteristics and prognosis of fumarate hydratase-deficient uterine leiomyoma (FH-dUL).Methods:Clinical data and follow-up information for 117 patients with FH-dUL diagnosed through surgical pathology and immunohistochemistry in the First Affiliated Hospital of Nanjing Medical University from January 2020 to December 2024, were collected. A control group of 130 patients with common uterine leiomyomas was also included. The differences between the two groups in clinical, imaging, and pathological characteristics were compared. Additionally, recurrence rates, fertility outcomes for FH-dUL patients, and the incidence of renal cancer in FH germline mutation carriers were monitored.Results:(1) Comparison of clinicopathological characteristics: the median age of 117 FH-dUL patients was 35 years, and the median age at first diagnosis of uterine leiomyomas was 29 years, both significantly younger than the control group (41 and 36 years; both P<0.01). The FH-dUL group showed significantly higher incidences of uterine myomectomy, multiple leiomyomas, diffusion restriction on pelvic magnetic resonance imaging diffusion weighted imaging, and typical pathological features (candelabra-like vessels, bizarre nuclei, cytoplasmic eosinophilic globules, perinuclear halo, cellular atypia) and higher ultrasound blood flow score (all P<0.05). Of the 30 FH-dUL patients who underwent genetic testing, 9 had germline mutations, 3 had somatic mutations, and 6 had mutations of unclear origin. Among the 9 FH gene germline mutation patients, 2 had already developed renal cell carcinoma. (2) Recurrence analysis: among the 56 patients who underwent uterine myomectomy, 22 (39.3%, 22/56) experienced recurrence during follow-up, compared to 12 (21.8%, 12/55) of the 55 patients in the control group, the difference between the two groups was statistically significant ( P=0.046). Multivariate binary logistic regression analysis showed that cellular leiomyomas ( OR=9.489, 95% CI: 1.740-51.755; P=0.009) and multiple uterine leiomyomas ( OR=10.709, 95% CI: 1.354-84.683; P=0.025) were significant risk factors for recurrence in FH-dUL. (3) Fertility analysis: among the 66 FH-dUL patients who underwent fertility-preserving surgery, 16 had the intention to have fertility desire, only 2 (2/16) completed their fertility plans during follow-up. Conclusions:Clinicopathological features and imaging features help to differentiate FH-dUL from common type uterine fibroids, but lack specificity, and the diagnosis of FH-dUL is based on immunohistochemistry. The recurrence rate after resection of FH-dUL is high, and cellular and multiple leiomyomas are important predictors of recurrence. It is crucial to perform genetic testing, genetic counseling, drug treatment to prevent recurrence, fertility guidance, and long-term comprehensive management after surgery for FH-dUL management.

Objective:To compare and analyze the differences in clinical and pathological features of uterine smooth muscle tumor of uncertain malignant potential (STUMP), common uterine leiomyoma (UL), and cellular uterine leiomyoma (CUL), and to identify biomarkers for predicting STUMP recurrence.Methods:A total of 72 cases of STUMP patients (STUMP group) treated at the First Affiliated Hospital of Nanjing Medical University and the First Affiliated Hospital of Soochow University were collected from June 2015 to March 2024. Additionally, 72 cases of UL and 72 cases of CUL (UL group and CUL group) in the same period were collected as controls. The clinical and pathological features of the three groups were compared, and the recurrence rates and related factors affecting STUMP recurrence were analyzed.Results:(1) Comparison of clinical and pathological features: there were statistically significant differences in age, history of myomectomy, and preoperative serum lactate dehydrogenase (LDH) levels among STUMP, UL, and CUL groups (all P<0.05). STUMP group were significantly older than UL group ( P<0.05). The proportions of STUMP group with a history of myomectomy and elevated preoperative serum LDH levels were significantly higher than those in UL and CUL groups (all P<0.05). On ultrasound, 16 cases of STUMP patients (22%, 16/72), 2 cases of UL patients (3%, 2/72), and 8 cases of CUL patients (11%, 18/72) had unclear fibroid borders, with significant differences between the three groups ( χ2=12.94, P=0.002), with STUMP group significantly higher than UL group ( P<0.05). Regarding immunohistochemistry, the proportion of p16 positivity, p53 mutations, and nuclear antigen associated with cell proliferation (Ki-67) >10% were significantly higher in STUMP group compared to UL group ( P<0.05). In terms of surgical approach, 52 cases of STUMP patients (72%, 52/72) underwent hysterectomy, compared to 27 cases of UL patients (38%, 27/72) and 38 cases of CUL patients (53%, 38/72), with a significant difference between the three groups ( χ2=17.89, P=0.001). The proportion of patients who underwent myomectomy was significantly lower in STUMP group compared to UL and CUL groups (both P<0.05). Among the 20 cases of STUMP patients who underwent myomectomy, 6 patients had a subsequent total hysterectomy after being diagnosed with STUMP. (2) Comparison of recurrence: the median follow-up time for the STUMP, UL, and CUL groups was 38, 12, and 29 months, respectively. During the follow-up period, 3 cases (6%, 3/53) in STUMP group, 4 cases (7%, 4/55) in UL group, and 8 cases (13%, 8/62) in CUL group had recurrence, with no significant differences between the three groups ( χ2=1.91, P=0.411). Among the 3 cases of STUMP patients with recurrence (in the pelvic cavity, liver, and abdominal wall), 2 cases had STUMP pathology on recurrence, and 1 case progressed to well-differentiated uterine leiomyosarcoma. (3) Related factors affecting STUMP recurrence: when comparing preoperative body mass index, serum LDH levels, and Ki-67 positivity ≥30% between recurrent and non-recurrent patients, significant differences were observed (all P<0.05). Univariate logistic regression analysis showed that Ki-67 positivity ≥30% was a significant risk factor for STUMP recurrence ( OR=24.67, 95% CI: 1.70-357.36, P=0.019). Conclusions:Factors such as age, history of myomectomy, preoperative serum LDH levels, and ultrasound findings of unclear fibroid borders are helpful for distinguishing STUMP from UL and CUL. Elevated preoperative serum LDH levels and Ki-67 positivity ≥30% have predictive value for STUMP recurrence. Active postoperative follow-up is essential, whether STUMP patients undergo myomectomy or hysterectomy.

This study was aimed at understanding the trends in,and scope of,severe fever with thrombocytopenia syndrome(SFTS)in Nanjing,analyzing the spatial distribution pattern,detecting high incidence cluster areas and key popula-tions,and scientifically guiding prevention and control strategies and measures.We obtained data on SFTS cases from 2010 to 2023 in Nanjing from the China Disease Control and Prevention Information System,and described the time,popu-lation,and spatial distribution characteristics.We used joinpoint regression to calculate the annual percentage change(APC)in incidence,then used FleXScan spatial clustering scan analysis to explore spatial clustering areas at the street level.A total of 507 SFTS cases were reported from 2010 to 2023 in Nanjing.The APC was 31.8％(95％CI:22.5％-41.9％,P＜0.001),and the reported incidence in 2023 was 1.42/100 000(134 cases).The seasonal indices from May to August were 2.7,2.1,3.0,and 1.3,respectively,accounting for 76.1％of the total cases.The median age was 66(IQR:55,73)years,which gradually increased from 59 years in 2010-2011 to 68 in 2022-2023(P＜0.001);94.1％of cases were in individuals 45 years or older.Farmers,homemakers/unemployed individuals,and retirees accounted for 90.1％.The epidemic area increased from 11 streets in four districts in 2010-2011 to 58 streets in 11 dis-tricts in 2022-2023.Except for 2012-2013,global spatial autocorrelation analysis showed positive Moran's I values(0.224-0.526,P＜0.001),and FlexScan scan indicated that several streets in Lishui District and Jiangning District were the most likely clusters.Four streets in Pukou District were the secondary clusters from 2018 to 2023,and three streets in Luhe District in 2022-2023 were the secondary clusters(all P＜0.05).The reported incidence of SFTS in Nanjing showed a rapid upward trend,with spread of epidemic areas.The spatial distribution pattern was clustered.Strengthened training in diagnosis and treatment technology and detection ability of medical institutions,surveillance in high-incidence areas,tracing of case flow,and health education of tick and disease prevention knowledge are recommended.

Objective:To prepare mesenchymal stem cells with antioxidant capacity (AO-MSC ) from human umbilical cords and evaluate its cell biological properties.Methods:In control group,mesenchymal stem cells (MSC) were isolated by digesting human umbilical cord Wharton's Jelly tissues with 0.2％ collagenase Ⅱ,and the released cells were collected and cultured in an animal serum-free culture medium.In AO-MSC group,incompletely collagenase Ⅱ-digested tissue debris were allowed to adhere to flusk flat bottoms and the AO-MSC was harvested by adherent culture. The conventional digestion and culture method was used as control.MSC colony forming ability was evaluated by fibroblast colony forming assay (CFU-F).MSC proliferative capacity was evaluated by CCK-8 assay.The MSC surface markers were detected by using flow cytometry and immunofluorescence staining.The adipogenic and osteogenic capacity of MSC was evaluated by multi-differentiation in vitro,and the mRNA expression of genes that control adipogenic and osteogenic differentiation was detected by real-time fluorescence quantitative PCR (RT-qPCR );Moreover,the mRNA expression of antioxidant substances such as SOD-1,GSH,GAT,and NQO1 in MSC was also evaluated by RT-qPCR.Results:The AO-MSC isolated by this strategy reached a confluence of 80％-90％ at around 18 days and grew in a swirling pattern.Flow cytometry and immunofluorescence staining assays showed that CD73,CD29,CD105,CD90 were highly expressed and CD31,CD45,HLA-DR were scarcely expressed in AO-MSC.AO-MSC exhibited stronger self-renewal and differentiation ability compared to MSC.However,the in vitro adipogenic-osteogenic capacity of MSC in the control group was stronger than that of AO-MSC.RT-qPCR assay showed that AO-MSC expressed higher mRNA levels of antioxidant substances compared to MSC.Conclusion:Human AO-MSC is successfully prepared from human umbilical cord without animal serum.

Objective To explore the efficacy and safety of recombinant human anti-severe acute respiratory syn-drome coronavirus 2(anti-SARS-CoV-2)monoclonal antibody injection(F61 injection)in the treatment of patients with coronavirus disease 2019(COVID-19)combined with renal damage.Methods Patients with COVID-19 and renal damage who visited the PLA General Hospital from January to February 2023 were selected.Subjects were randomly divided into two groups.Control group was treated with conventional anti-COVID-19 therapy,while trial group was treated with conventional anti-COVID-19 therapy combined with F61 injection.A 15-day follow-up was conducted after drug administration.Clinical symptoms,laboratory tests,electrocardiogram,and chest CT of pa-tients were performed to analyze the efficacy and safety of F61 injection.Results Twelve subjects(7 in trial group and 5 in control group)were included in study.Neither group had any clinical progression or death cases.The ave-rage time for negative conversion of nucleic acid of SARS-CoV-2 in control group and trial group were 3.2 days and 1.57 days(P=0.046),respectively.The scores of COVID-19 related target symptom in the trial group on the 3rd and 5th day after medication were both lower than those of the control group(both P＜0.05).According to the clinical staging and World Health Organization 10-point graded disease progression scale,both groups of subjects improved but didn't show statistical differences(P＞0.05).For safety,trial group didn't present any infusion-re-lated adverse event.Subjects in both groups demonstrated varying degrees of elevated blood glucose,elevated urine glucose,elevated urobilinogen,positive urine casts,and cardiac arrhythmia,but the differences were not statistica-lly significant(all P＞0.05).Conclusion F61 injection has initially demonstrated safety and clinical benefit in trea-ting patients with COVID-19 combined with renal damage.As the domestically produced drug,it has good clinical accessibility and may provide more options for clinical practice.

ObjectiveTo investigate the therapeutic effect of Dayuanyin on acute lung injury induced by H1N1 infection and decipher the potential mechanism. MethodThe constituents in Dayuanyin were analyzed by ultra-high performance liquid chromatography-quadrupole-exactive orbitrap mass spectrometry （UHPLC-Q-Exactive Orbitrap MS）. Forty-eight female BALB/c mice were randomized into normal， model， oseltamivir （19.5 mg·kg^-1）， and low-， medium-， and high-dose （2.73， 5.46， 10.92 g·kg^-1） Dayuanyin groups. The normal and model groups were administrated with deionized water by gavage， and the other groups were administrated with the corresponding drugs by gavage. On day 3 of drug administration， the normal group received nasal inhalation of normal saline， and the other groups were inoculated intranasally with A/RP/8/34 （H1N1） for the modeling of influenza virus infection. Mice were administrated with drugs continuously for 7 days and weighed daily. Sampling was performed 12 h after the last administration， and the lung tissue was weighed to calculate the lung index. Hematoxylin-eosin staining was performed to observe the pathological and morphological changes of the lung tissue and bronchi. The cytometric bead array （CBA） was used to measure the serum levels of interferon-gamma （IFN-γ）， C-X-C motif ligand 1 （CXCL1）， tumor necrosis factor-alpha （TNF-α）， chemokine ligand 2 （CCL2）， interleukin-12p70 （IL-12p70）， chemokine ligand 5 （CCL5）， interleukin-1β （IL-1β）， chemokine （C-X-C motif） ligand 10 （CXCL10）， granulocyte-macrophage colony-stimulating factor （GM-CSF）， interleukin-10 （IL-10）， interferon-beta （IFN-β）， interferon-alpha （IFN-α）， and interleukin-6 （IL-6）. According to the results of mass spectrometry and network pharmacology， we analyzed the mechanism of Dayuanyin in treating acute lung injury caused by H1N1. The protein levels of extracellular signal-regulated kinase 1/2 （ERK1/2）， p38 mitogen-activated protein kinase （p38 MAPK）， nuclear factor-kappa B （NF-κB）， and their phosphorylated forms were determined by Western blot. The mRNA levels of myeloid differentiation factor 88 （MyD88）， Toll-like receptor 3 （TLR3）， Toll-like receptor 7 （TLR7）， and Toll-like receptor 8 （TLR8） in the lung tissue were measured by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. ResultA total of 57 compounds， including paeoniflorin and baicalein， were detected in Dayuanyin. Compared with the normal group， the model group showed decreased body weight （P<0.01）， lung edema and hemorrhage， increased lung index （P<0.01）， and elevated levels of IFN-γ， IL-12p70， CCL5， IL-1β， CXCL10， GM-CSF， IFN-β， and IL-6 （P<0.01）. Compared with the model group， Dayuanyin attenuated alveolar wall thickening， capillary congestion， and immune cell infiltration， reduced the alterations in body weight and lung index （P<0.01）， and down-regulated the protein levels of IFN-γ， IL-12p70， CCL5， IL-1β， CXCL10， GM-CSF， IFN-β， and IL-6 （P<0.01）. A total of 57 key genes were predicted by network pharmacological analysis， of which the MAPK signaling pathway was the main target signaling pathway. Compared with the normal group， the model group showed up-regulation in the protein levels of phosphorylation （p）-ERK1/2， p-p38 MAPK， and p-NF-κB （P<0.01） and the mRNA levels of TLR7， TLR8， MyD88， and TLR3 （P<0.05， P<0.01）. Compared with the model group， Dayuanyin lowered the phosphorylation levels of ERK1/2， p38 MAPK， and NF-κB p65 in a dose-dependent manner （P<0.01） and down-regulated the mRNA levels of TLR3， TLR7， TLR8， and MyD88 （P<0.01）. ConclusionDayuanyin can prevent and control H1N1 infection-induced acute lung injury by inhibiting the TLR/MAPK/NF-κB signaling pathway.

Objective:To compare and analyze the differences in clinical and pathological features of uterine smooth muscle tumor of uncertain malignant potential (STUMP), common uterine leiomyoma (UL), and cellular uterine leiomyoma (CUL), and to identify biomarkers for predicting STUMP recurrence.Methods:A total of 72 cases of STUMP patients (STUMP group) treated at the First Affiliated Hospital of Nanjing Medical University and the First Affiliated Hospital of Soochow University were collected from June 2015 to March 2024. Additionally, 72 cases of UL and 72 cases of CUL (UL group and CUL group) in the same period were collected as controls. The clinical and pathological features of the three groups were compared, and the recurrence rates and related factors affecting STUMP recurrence were analyzed.Results:(1) Comparison of clinical and pathological features: there were statistically significant differences in age, history of myomectomy, and preoperative serum lactate dehydrogenase (LDH) levels among STUMP, UL, and CUL groups (all P<0.05). STUMP group were significantly older than UL group ( P<0.05). The proportions of STUMP group with a history of myomectomy and elevated preoperative serum LDH levels were significantly higher than those in UL and CUL groups (all P<0.05). On ultrasound, 16 cases of STUMP patients (22%, 16/72), 2 cases of UL patients (3%, 2/72), and 8 cases of CUL patients (11%, 18/72) had unclear fibroid borders, with significant differences between the three groups ( χ2=12.94, P=0.002), with STUMP group significantly higher than UL group ( P<0.05). Regarding immunohistochemistry, the proportion of p16 positivity, p53 mutations, and nuclear antigen associated with cell proliferation (Ki-67) >10% were significantly higher in STUMP group compared to UL group ( P<0.05). In terms of surgical approach, 52 cases of STUMP patients (72%, 52/72) underwent hysterectomy, compared to 27 cases of UL patients (38%, 27/72) and 38 cases of CUL patients (53%, 38/72), with a significant difference between the three groups ( χ2=17.89, P=0.001). The proportion of patients who underwent myomectomy was significantly lower in STUMP group compared to UL and CUL groups (both P<0.05). Among the 20 cases of STUMP patients who underwent myomectomy, 6 patients had a subsequent total hysterectomy after being diagnosed with STUMP. (2) Comparison of recurrence: the median follow-up time for the STUMP, UL, and CUL groups was 38, 12, and 29 months, respectively. During the follow-up period, 3 cases (6%, 3/53) in STUMP group, 4 cases (7%, 4/55) in UL group, and 8 cases (13%, 8/62) in CUL group had recurrence, with no significant differences between the three groups ( χ2=1.91, P=0.411). Among the 3 cases of STUMP patients with recurrence (in the pelvic cavity, liver, and abdominal wall), 2 cases had STUMP pathology on recurrence, and 1 case progressed to well-differentiated uterine leiomyosarcoma. (3) Related factors affecting STUMP recurrence: when comparing preoperative body mass index, serum LDH levels, and Ki-67 positivity ≥30% between recurrent and non-recurrent patients, significant differences were observed (all P<0.05). Univariate logistic regression analysis showed that Ki-67 positivity ≥30% was a significant risk factor for STUMP recurrence ( OR=24.67, 95% CI: 1.70-357.36, P=0.019). Conclusions:Factors such as age, history of myomectomy, preoperative serum LDH levels, and ultrasound findings of unclear fibroid borders are helpful for distinguishing STUMP from UL and CUL. Elevated preoperative serum LDH levels and Ki-67 positivity ≥30% have predictive value for STUMP recurrence. Active postoperative follow-up is essential, whether STUMP patients undergo myomectomy or hysterectomy.

OBJECTIVE: To establish an intestinal organoid model that mimic acute graft versus host disease (aGVHD) caused intestinal injuries by using aGVHD murine model serum and organoid culture system, and explore the changes of aGVHD intestine in vitro by advantage of organoid technology. METHODS: 20-22 g female C57BL/6 mice and 20-22 g female BALB/c mice were used as donors and recipients for bone marrow transplantation, respectively. Within 4-6 h after receiving a lethal dose (8.0 Gy) of γ ray total body irradiation, a total of 0.25 ml of murine derived bone marrow cells (1×10⁷/mice, n=20) and spleen nucleated cells (5×10⁶/mice, n=20) was infused to establish a mouse model of aGVHD (n=20). The aGVHD mice were anesthetized at the 7th day after transplantation, and the veinal blood was harvested by removing the eyeballs, and the serum was collected by centrifugation. The small intestinal crypts of healthy C57BL/6 mice were harvested and cultivated in 3D culture system that maintaining the growth and proliferation of intestinal stem cells in vitro. In our experiment, 5%, 10%, 20% proportions of aGVHD serum were respectively added into the organoid culture system for 3 days. The formation of small intestinal organoids were observed under an inverted microscope and the morphological characteristics of intestinal organoids in each groups were analyzed. For further evaluation, the aGVHD intestinal organoids were harvested and their pathological changes were observed. Combined with HE staining, intestinal organ morphology evaluation was performed. Combined with Alcian Blue staining, the secretion function of aGVHD intestinal organoids was observed. The distribution and changes of Lgr5⁺ and Clu⁺ intestinal stem cells in intestinal organoids were analyzed under the conditions of 5%, 10% and 20% serum concentrations by immunohistochemical stainings. RESULTS: The results of HE staining showed that the integrity of intestinal organoids in the 5% concentration serum group was better than that in the 10% and 20% groups. The 5% concentration serum group showed the highest number of organoids, the highest germination rate and the lowest pathological score among experimental groups, while the 20% group exhibited severe morphological destruction and almost no germination was observed, and the pathological score was the highest among all groups(t=3.668, 4.334,5.309,P<0.05). The results of Alican blue staining showed that the secretion function of intestinal organoids in serum culture of aGVHD in the 20% group was weaker than that of the 5% group and 10% of the organoids, and there was almost no goblet cells, and mucus was stainned in the 20% aGVHD serum group. The immunohistochemical results showed that the number of Lgr5⁺ cells of intestinal organoids in the 5% group was more than that of the intestinal organoids in the 10% aGVHD serum group and 20% aGVHD serum group. Almost no Clu⁺ cells were observed in the 5% group. The Lgr5⁺ cells in the 20% group were seriously injuried and can not be observed. The proportion of Clu⁺ cells in the 20% group significantly increased. CONCLUSION The concentration of aGVHD serum in the culture system can affect the number and secretion function of intestinal organoids as well as the number of intestinal stem cells in organoids. The higher the serum concentration, the greater the risk of organoid injury, which reveal the characteristics of the formation and functional change of aGVHD intestinal organoids, and provide a novel tool for the study of intestinal injury in aGVHD.
Mice ; Female ; Animals ; Mice, Inbred C57BL ; Bone Marrow Transplantation ; Graft vs Host Disease ; Stem Cells ; Organoids