UV/Vis-based process analytical technology to improve monoclonal antibody and host cell protein separation

Process analytical technology(PAT) is gaining more interest in the biomanufacturing industry because of its potential to improve operational control and compliance through real-time quality assurance.Currently, biopharmaceutical producers mainly monitor chromatographic processes with ultraviolet/visible(UV/Vis) absorbance. However, this measurement has a very limited correlation with purity and quantity. The current study aims to determine the concentration of monoclonal antibody(mAb) and host cell proteins(HCPs) using a build-in UV/Vis monitoring during Protein A affinity chromatography and to optimize the separation conditions for high purity of mAb and minimizing the HCPs content. The eluate was analyzed through in-line UV/Vis at 280 and 410 nm, representing mAb and HCPs concentration,respectively. Each 0.1 column volume(CV) fraction of UV/Vis chromatogram peak area were calculated,and different separation conditions were then compared. The optimum conditions of mAb separation were found as 12 CV loading, elution at pH 3.5, and starting the collection at 0.5 CV point, resulting in high m Ab recovery of 95.92% and additional removal of 49.98% of HCP comparing with whole elution pool. This study concluded that UV/Vis-based in-line monitoring at 280 and 410 nm showed a high potential to optimize and real-time control Protein A affinity chromatography for mAb purification from HCPs.

Host cell protein quantification workflow using optimized standards combined with data-independent acquisition mass spectrometry

Monitoring of host cell proteins(HCPs)during the manufacturing of monoclonal antibodies(mAb)has become a critical requirement to provide effective and safe drug products.Enzyme-linked immunosorbent assays are still the gold standard methods for the quantification of protein impurities.However,this technique has several limitations and does,among others,not enable the precise identification of proteins.In this context,mass spectrometry(MS)became an alternative and orthogonal method that delivers qualitative and quantitative information on all identified HCPs.However,in order to be routinely implemented in biopharmaceutical companies,liquid chromatography-MS based methods still need to be standardized to provide highest sensitivity and robust and accurate quantification.Here,we present a promising MS-based analytical workflow coupling the use of an innovative quantification standard,the HCP Profiler solution,with a spectral library-based data-independent acquisition(DIA)method and strict data validation criteria.The performances of the HCP Profiler solution were compared to more conventional standard protein spikes and the DIA approach was benchmarked against a classical datadependent acquisition on a series of samples produced at various stages of the manufacturing process.While we also explored spectral library-free DIA interpretation,the spectral library-based approach still showed highest accuracy and reproducibility(coefficients of variation<10%)with a sensitivity down to the sub-ng/mg mAb level.Thus,this workflow is today mature to be used as a robust and straightforward method to support mAb manufacturing process developments and drug products quality control.

Detection and quantitation of host cell proteins in monoclonal antibody drug products using automated sample preparation and data-independent acquisition LC-MS/MS

Ensuring the removal of host cell proteins(HCPs) during downstream processing of recombinant proteins such as monoclonal antibodies(m Abs) remains a challenge.Since residual HCPs might affect product stability or safety,constant monitoring is required to demonstrate their removal to be below the regulatory accepted level of 100 ng/mg.The current standard analytical approach for this procedure is based on ELISA;however,this approach only measures the overall HCP content.Therefore,the use of orthogonal methods,such as liquid chromatography-mass spectrometry(LC-MS),has been established,as it facilitates the quantitation of total HCPs as well as the identification and quantitation of the individual HCPs present.In the present study,a workflow for HCP detection and quantitation using an automated magnetic bead-based sample preparation,in combination with a data-independent acquisition(DIA) LC-MS analysis,was established.Employing the same instrumental setup commonly used for peptide mapping analysis of m Abs allows for its quick and easy implementation into pre-existing workflows,avoiding the need for dedicated instrumentation or personnel.Thereby,quantitation of HCPs over a broad dynamic range was enabled to allow monitoring of problematic HCPs or to track changes upon altered bioprocessing conditions.

Quantitative analysis of host cells growing into canine homograft valved aortic and pulmonary artery

Background Cryopreserved conduit valved homografts （CVH） have been widely used in surgical treatment of cardiac disease. This study aimed to determine the extent of host cell ingrowth and the durability and immunogenicity of CVH,and to compare the performance of CVH stored at 4℃ and CVH cryopreserved in liquid nitrogen at -196℃.Methods Heterotopic transplants of canine CVH stored at 4℃ （n=14） and cryopreserved in liquid nitrogen （n=14） were made onto the abdominal aorta of recipient dogs. Animals were sacrificed at 7 and 15 days and at 1, 3, 6, 9, and 12months after transplantation to excise the implanted CVHs. Tissue DNA extraction and quantitative polymerase chain reaction （PCR） were performed to calculate the ratio of donor cells and host cells in the CVH. The tissue viability of CVH after implantation was analyzed by detecting alkaline fibroblast growth factor 2 （FGF-2） using immunohistochemical staining and by observation under transmission electron microscope and scanning electron microscope.Results All the animals survived and recovered well. There were few repopulating host cells （0.04-0.83%） in the implanted CVH at 7 or 15 days. The ratio of ingrowing host cells into the CVH continued rising after implantation and reached 40%-47% in the 12th month postoperation. Histology, transmission electron microscopy and FGF-2immunohistochemical staining indicated that fibroblasts and the host＇s endothelial cells were the main cellular elements invading the CVH. There were no significant differences in results between CVH stored at 4℃ and CVH cryopreserved in liquid nitrogen.Conclusions Host cells growing into CVH are very important for maintaining the long-term structure and function of the implanted CVH. There is no significant difference between CVH storing at 4℃ or in liquid nitrogen in regard to the ingrowth of host cells or of morphologic features after CVH allografting.

Virus/host cell interactions:From structure to medical implication

The first event in viral infection is the attachment of a virus to specific receptors on the host cell surface. This will trigger conformational changes of the viral surface protein. For

Cell-based therapies for neural replacement strategies in stroke-related neurodegeneration： neurophysiological insights into stem progenitor cell neurogenesis within a host environment

The restricted neurogenesis limits the brain ability to overcome neuronal cell death following ischemic lesion：Failure of the damaged brain to regenerate following cerebral ischemia results in functional deficits those are most often irreversible and can further deteriorate,causing mortality and severe disability,progressive memory loss and cognitive impairments,known as dementia.

Umbilical Cord Blood-derived Mesenchymal Stem Cells Ameliorate Graft-Versus-Host Disease Following Allogeneic Hematopoietic Stem Cell Transplantation through Multiple Immunoregulations

Summary： Although mesenchymal stem cells （MSCs） are increasingly used to treat graft-versus-host disease （GVHD）, their immune regulatory mechanism in the process is elusive. The present study aimed to investigate the curative effect of third-party umbilical cord blood-derived human MSCs （UCB-hMSCs） on GVHD patients after allogeneic hematopoietic stem cell transplantation （allo-HSCT） and their immune regulatory mechanism. Twenty-four refractory GVHD patients after allo-HSCT were treated with UCB-hMSCs. Immune cells including T lymphocyte subsets, NK ceils, Treg cells and dendritic cells （DCs） and cytokines including interleukin-17 （IL-17） and tumor necrosis factor-alpha （TNF-α） were monitored before and after MSCs transfusion. The results showed that the symptoms of GVHD were alleviated significantly without increased relapse of primary disease and transplant-related complications after MSCs transfusion. The number of CD3^＋, CD3＋CD4^＋ and CD3＋CD8^＋ cells decreased significantly, and that of NK cells remained unchanged, whereas the number of CD4^＋ and CD8^＋ Tregs increased and reached a peak at 4 weeks; the number of mature DCs, and the levels of TNF-α and IL-17 decreased and reached a trough at 2 weeks. It was concluded that MSCs ameliorate GVHD and spare GVL effect via immunoregulations.

Immune Regulatory Cell Biology and Clinical Applications to Prevent or Treat Acute Graft-Versus-Host Disease

The most common approaches to prevent and treat graft-versus-host disease (GVHD) are intended to deplete or suppress the T cells capable of mediating or supporting alloresponses;however, this renders the recipients functionally T cell deficient and hence highly susceptible to infections and tumor recurrence. Depletion is often accomplished through the use of broadly reactive antibodies, while functional impairment is typically achieved by pharmacological agents that require long-term administration (usually six months or more), have significant side effects, and may not result in tolerance (i.e., nonresponsiveness) of donor T cells to conditioning regimen-resistant host alloantigen-bearing cells. As our knowledge of immune system homeostasis has increased, cell populations with immune regulatory function have been identified and characterized. Although such cell populations are typically present in low frequencies, methods to isolate and expand these cells have permitted their supplementation to the donor graft or infusion late post-transplant in order to stifle GVHD. This review discusses the biology and preclinical proof of concept of GVHD models, along with GVHD outcomes that focus exclusively on immune regulatory cell therapies that have progressed to clinical testing.

OMISSION OF DAY +11 METHOTREXATE DOES NOT APPEAR TO INFLUENCE INCIDENCE AND SEVERITY OF GRAFT-VERSUS- HOST DISEASE AFTER ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION

Objective: To explore the influence of omission of the day +11 dose of methotrexate (MIX) on the incidence and severity of graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Methods: From April 1997 to October 2002, 80 leukemia patients (46 men and 34 women aged from 12 to 56 years with a median age of 35) underwent allo-HSCT at our BMT unit. Among them, 58 patients received grafts from HLA-identical siblings, 8 from HLA one major antigen mismatched siblings and 14 from HLA-matched unrelated donors. All patients received a modified cyclosporine and short-course MTX regimen for GVHD prophylaxis, which included MTX 15 mg on day +1, and 10 mg on days +3 and +6 (MTX day +11 dose omitted) and cyclosporine given daily. Results: The overall incidence of grade I~IV acute GVHD was 57.5% (46/80 patients), with grade II~IV acute GVHD in 28 patients (35%) and grade III~IV acute GVHD in 7 patients (8.8%). Among 58 patients receiving grafts from HLA-identical siblings, 24 patients developed grade I~IV acute GVHD (41.4%), with grade II~IV acute GVHD in 13 patients (22.4%) and grade III~IV acute GVHD in 4 patients (6.9%). 2l out of 22 patients receiving grafts from HLA one major antigen mismatched siblings and HLA-matched unrelated donors developed grade I~IV acute GVHD (95.5%), with grade II~IV acute GVHD in 14 patients (63.6%) and grade III~IV acute GVHD in 3 patients (13.6%). Chronic GVHD occurred in 38 out of 56 evaluable patients (67.9%), with extensive form in 15 patients (26.8%) and limited form in 23 patients (41.1%). With a median follow-up of 960 days (range 180~1980 days), the probability of leukemia-free survival at 3 years was 61.3% for all patients. Conclusion: Our results suggest that the day +11 MTX can be omitted without a major deleterious effect on the incidence and severity of graft-versus-host disease after HLA-identical sibling transplantation as well as HLA one major antigen mismatched sibling and HLA-matched unrelated donor transplantation.

Expressions of Tissue Factor and Tissue Factor Pathway Inhibitor in Patients with Acute Graft-versus-host Disease after Allogeneic Hematopoietic Stem Cell Transplantation

This study examined the expressions of human serum tissue factor （TF） and tissue factor pathway inhibitor （TFPI） in patients with acute graft-versus-host disease （aGVHD） after allogeneic hematopoietic stem cell transplantation （allo-HSCT） and their clinical significance. The serum TF and TFPI levels were detected by ELISA in 28 allo-HSCT recipients before and after the transplanta-tion and the changes of TF and TFPI levels were dynamically monitored at different phases of the disease. No significant differences in the serum TF and TFPI levels were found in allo-HSCT recipi-ents in the absence of aGVHD or with gradeⅠaGVHD before and after the transplantation. The lev-els of serum TF and TFPI were substantially increased in the patients with gradeⅡ aGVHD at the peak of aGVHD （P〈0.05） and they were even higher in the patients with grade Ⅲ–Ⅳ aGVHD （P〈0.01）. When the conditions became stable after treatment with immunosuppressive agents, the serum TFPI level was decreased to the baseline level （P〉0.05） and the TF level was lowered but still higher than the baseline level （P〈0.05）. It was concluded that the levels of serum TF and TFPI were increased significantly in the patients with grade Ⅱ–Ⅳ aGVHD after allo-HSCT and decreased markedly after the treatment. Monitoring the levels of serum TF and TFPI in the patients with allo-HSCT is important to predict the occurrence, outcome and prognosis of aGVHD.

Ruxolitinib add-on in corticosteroid-refractory graft-vs-host disease after allogeneic stem cell transplantation:Results from a retrospective study on 38 Chinese patients

BACKGROUND Graft-vs-host disease (GVHD) is a major cause of mortality after allogeneic hematopoietic stem cell transplantation.Some patients have steroid-refractory(SR) GVHD.AIM To evaluate the effect and safety of ruxolitinib add-on in the treatment of patients with SR acute (a) and chronic (c) GVHD.METHODS We retrospectively analyzed 38 patients administered ruxolitinib add-on to standard immunosuppressive therapy for SR-aGVHD or SR-cGVHD following allogeneic hematopoietic stem cell transplantation.Ruxolitinib was administered5-10 mg/d depending on disease severity,patient status,and the use of antifungal drugs.Overall response rate,time to best response,malignancy relapse rate,infection rate,and treatment-related adverse events were assessed.RESULTS The analysis included 10 patients with SR-aGVHD (gradeⅢ/Ⅳ,n=9) and 28patients with SR-cGVHD (moderate/severe,n=24).For the SR-aGVHD and SRcGVHD groups,respectively:Median number of previous GVHD therapies was 2(range:1-3) and 2 (1-4);median follow-up was 2.5 (1.5-4) and 5 (1.5-10) mo;median time to best response was 1 (0.5-2.5) and 3 (1-9.5) mo;and overall response rate was 100%(complete response:80%) and 82.1%(complete response:10.7%) with a response observed in all GVHD-affected organs.The malignancy relapse rates for the SR-aGVHD and SR-cGVHD groups were 10.0%and 10.7%,respectively.Reactivation rates for cytomegalovirus,Epstein-Barr virus,and varicella-zoster virus,respectively,were 30.0%,10.0%,and 0%for the SR-aGVHD group and 0%,14.3%,and 7.1%for the SR-cGVHD group.CONCLUSION Ruxolitinib add-on was effective and safe as salvage therapy for SR-GVHD.

生物质谱在生物制品宿主细胞蛋白残留分析中的应用

宿主细胞蛋白(host cell proteins,HCPs)残留影响生物制品的质量和安全,是生物制品生产的关键质控要素。目前,HCPs残留的主要质控方法是酶联免疫吸附试验(ELISA),但该方法的准确性高度依赖于抗体的特异性,且无法获得HCPs的种类及其含量分布信息,需要使用正交方法全面监测。而生物质谱技术无需依赖抗体即可实现HCPs的定性和定量分析,已逐渐成为除ELISA法外的主要分析表征方法,但质谱技术存在缺乏统一的操作流程和验证标准、高丰度药物信号抑制以及高昂的仪器成本等问题。本文总结了质谱法在生物制品HCPs分析中的工作流程及其应用进展,详细阐述了流程中涉及的样品准备、液相色谱分离、质谱数据采集、质谱数据分析和报告的开发原则和关键方法参数,并对未来的研究方向及挑战进行展望。

根结线虫诱导的巨型细胞及其形成机制

根结线虫(Meloidogyne spp.)是一种专性寄生线虫,具有分布广泛、寄主繁多和危害严重的特点。每年给农作物造成巨大的经济损失,是危害农作物最严重的线虫之一。侵染植物时,根结线虫会诱导根尖部位的维管束细胞重新分化形成多核的巨型细胞,将其作为线虫的取食位点和线虫生长发育过程中营养物质的唯一来源。因此,巨型细胞的形成和维持对线虫的生长和繁殖至关重要。本文从根结线虫诱导寄主植物巨型细胞的形成及调控机制,重点介绍了巨型细胞的结构特征、巨型细胞的细胞周期变化、营养物质运输、激素调节,以及寄主植物的识别和防御和表达模式,并探讨了根结线虫效应蛋白在巨型细胞形成过程中的功能。通过对巨型细胞形成和调控机制及线虫-植物相互分子机制等多层次的研究进展进行解析,揭示根结线虫的致病机理,以期为后续研究根结线虫防治创新性策略提供启发和思路。

确定性筛选设计在Purcise Q膜纯化抗体的应用

目的:通过确定性筛选设计(DSD)方法建立单抗的Purcise Q膜阴离子交换工艺,进一步降低残留宿主细胞蛋白(HCP)含量。方法:将亲和层析洗脱过滤液作为样品,进行Purcise Q膜层析,选取上样pH、上样电导、载量、流速、峰收集条件5个因子为输入因子,以回收率、HCP去除率、多聚体含量为输出响应值,使用确定性筛选设计DOE模型进行实验。结果:在实验参数范围内,多聚体含量没有明显的变化,HCP含量显著降低。上样电导和载量显著影响HCP去除率,而载量、流速和峰收集条件则显著影响层析回收率。利用JMP的模拟实验功能获得了稳健工艺参数组合(上样电导3 ms/cm、载量300 g/L、流速25 MV/min、峰收集50 mAU/mm),进一步利用决策树机器学习方法自动获得设计空间(上样电导3.0~3.6 ms/cm、载量300~460 g/L、流速5~25 MV/min、峰收集50~180 mAU/mm)。结论:确定性筛选设计实验方法适用于膜层析的工艺开发,能够一段式快速获得稳健工艺参数组合和设计空间,对于工艺放大、工艺转移和降低生产风险极具指导意义。

细菌外囊泡的研究进展

细菌外囊泡(bacterial extracellular vesicles,BEVs)是由细菌分泌的球形双层脂质纳米颗粒,包含一系列来自亲本细菌的物质。BEVs是细菌和环境之间的重要信息传递者,其在细菌的生存和环境适应中发挥着多种作用,而且还能与宿主细胞膜作用,改变宿主细胞信号传导途径,从而影响宿主细胞的生理功能和疾病的发生与发展。本文综述了BEVs的生物发生机制、生物学功能及其对宿主细胞调节的最新研究进展,期望对深入研究BEVs的生物学功能和致病机制有所启示,并能够为其用于临床早期诊断、预防和治疗等方面研究提供新思路。

猪流行性腹泻病毒核衣壳蛋白的研究进展

猪流行性腹泻病毒(PEDV)是猪流行性腹泻(PED)的病原,PEDV可导致猪的急性腹泻、脱水甚至死亡。PEDV最早在1971年被报道,如今,它已成为引起仔猪腹泻的最常见病原体之一,对我国养猪行业造成极大的经济损失。核衣壳(N)蛋白是PEDV主要的结构蛋白质之一。本文对PEDV N蛋白的核定位信号、生理功能、与宿主细胞互作和检测方法四个方面的研究进展综述,以期能对N蛋白的了解更够更加深入,并为PED防控提供参考作用。

脂质代谢和糖代谢在PRRSV感染宿主细胞中作用研究进展

猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)感染可引起母猪繁殖障碍、仔猪呼吸道疾病及公猪精液质量下降,给世界养猪业造成了巨大的经济损失。PRRSV不能自主复制,其生命周期的各个阶段均依赖于宿主的代谢系统。宿主细胞也可调节其代谢过程,以防止PRRSV复制和维持其正常生理功能。脂质代谢和糖代谢在PRRSV感染中均扮演了重要角色,PRRSV作为一种囊膜病毒对脂质代谢系统的依赖性较其他代谢系统更强。脂质参与了PRRSV生命周期的各个阶段,包括吸附、进入、复制、组装和释放,此外还与细胞炎症、免疫和凋亡有关。糖代谢也可干扰PRRSV的生命活动,从而促进或抑制PRRSV复制。文章综述了脂质代谢中脂肪酸、胆固醇、磷脂、脂滴和脂筏以及糖代谢中糖酵解和三羧酸循环在PRRSV感染宿主细胞中的作用,以期为阐明PRRSV的致病机制以及疫苗和抗PRRSV药物的研发提供基本理论依据。

异基因造血干细胞移植后迟发型出血性膀胱炎的危险因素分析

目的:分析异基因造血干细胞移植(allo-HSCT)后并发迟发型出血性膀胱炎(LOHC)的危险因素、LOHC发展为重度LOHC的危险因素及LOHC对生存的影响。方法:对2015年1月-2021年12月在重庆医科大学附属第一医院行allo-HSCT的300例患者的临床资料进行回顾性研究,选择可能影响allo-HSCT后LOHC发生的相关临床参数进行单因素和多因素分析,同时分析组间的总生存期(OS)和无进展生存期(PFS)差异。结果:多因素分析结果显示,患者年龄≤45岁(P=0.039)、强化预处理方案中包含氟达拉滨/克拉屈滨+阿糖胞苷(P=0.002)、移植后d 30白蛋白≤30 g/L(P=0.007)、CMV-DNA+(P=0.028)、移植前有真菌感染(P=0.026)、Ⅱ-Ⅳ度a GVHD的发生(P=0.006)是发生LOHC的独立危险因素;在已发生LOHC的移植患者中,LOHC发生的时间在移植后32 d内(P=0.008)、移植后d 30的白蛋白≤30 g/L(P=0.032)是发展为重度LOHC的独立危险因素。重度LOHC组的OS率显著低于未发生LOHC组(P=0.041)。结论:对于年龄≤45岁、强化预处理或LOHC发生较早的移植患者,需要警惕发生LOHC或发展为重度LOHC,应早期做好防治;定期监测CMV-DNA、白蛋白水平,积极有效地抗病毒、抗真菌治疗及防治a GVHD是预防LOHC发生发展的有效措施。

The bacteriophage mu lysis system–A new mechanism of host lysis?

Bacteriophages are viruses that infect bacteria and can choose any one of the two alternative pathways for infection,i.e.,lysis or lysogeny.Phage lysis is one of the conventional biological processes required to spread infection from one bacterium to another.Our analysis suggests that in the paradigm bacteriophage Mu,six proteins might be involved in host cell lysis.Mu has a broad host range,and Mu-like phages were found in both Gram-negative and Gram-positive bacteria.An analysis of the genomes of Mu and Mu-like phages could be useful in elucidating the lysis mechanism in this group of phages.A detailed review of the various mechanisms of phage lysis and different proteins associated with the process will help researchers understand the phage biology and their life cycle in different bacteria.The recent increase in the number of multidrug-resistant(MDR)strains of bacteria and the usual long-term nature of new drug development has encouraged scientists to look for alternative strategies like phage therapy and the discovery of new lysis mechanisms.Understanding the lysis mechanism in the Mu-like phages could be exploited to develop alternative therapeutics to kill drug-resistant pathogenic bacteria.In this review article,we have analyzed the phage Mu-mediated host lysis system,which is unknown till now,and our analysis indicates a possibility of the existence of a new lysis mechanism operating in Mu.

黄芩汤调控肠道菌群治疗小鼠肠道急性移植物抗宿主病的机制

背景:肠道急性移植物抗宿主病是异基因造血干细胞移植后最凶险的并发症之一,具有较高的致死率,如何通过应用中药改善肠道炎症、调节自噬以治疗肠道急性移植物抗宿主病是当下值得研究的问题。目的:探讨黄芩汤调控肠道菌群治疗肠道急性移植物抗宿主病的机制。方法:CB6F1小鼠经总剂量8 Gy的60Co X射线照射后通过尾静脉输入Balb/c H-2d小鼠的单个核细胞悬液(骨髓+脾)制备急性移植物抗宿主病模型,随机分为模型组及黄芩汤高、中、低剂量组。造模后分别给予不同剂量黄芩汤或等体积生理盐水连续灌胃14 d,进行临床急性移植物抗宿主病评分并记录生存时间,取小肠组织做苏木精-伊红染色行小肠黏膜病理分级评分。使用16S rDNA测序检测各组小鼠肠道菌群,行免疫荧光、免疫组化染色、PCR等检测自噬相关标志物的表达。结果与结论:(1)与模型组相比,黄芩汤中、高剂量组小鼠存活时间显著延长(P<0.01),临床急性移植物抗宿主病评分显著降低(P<0.01),小肠黏膜病理分级评分显著降低(P<0.01),小肠组织炎症因子肿瘤坏死因子α、白细胞介素1β、白细胞介素6水平显著下降(P<0.01),小鼠小肠黏膜上皮结构完整性部分恢复;(2)肠道菌群研究发现,与模型组相比,黄芩汤中剂量组促炎菌株肠球菌显著减少(P<0.05),而有益菌如梭状芽孢杆菌及促自噬的红球菌显著增多(P<0.05);(3)与模型组相比,黄芩汤中剂量组的自噬标志物显著升高(P<0.05);透射电镜下,黄芩汤中剂量组自噬泡数量显著增多;(4)结果表明:黄芩汤显著降低条件性致病菌丰度和小肠组织炎症因子水平,并提高有益菌相对丰度,同时促进小肠黏膜自噬的表达,从而明显改善急性移植物抗宿主病小鼠肠道症状。