Collagen, a central component of the extracellular matrix (ECM), precisely regulates tissue mechanical properties and biological functions through hierarchical assembly, playing a vital role in maintaining homeostasis. However, the molecular mechanism of assembly remains poorly understood, limiting insights into tissue remodeling, aging, and ECM-related diseases. Here, we employ time-resolved cryo-electron microscopy to resolve two critical hierarchical intermediates in fibrillar collagen assembly, proposing the 3D collagen assembly pathway. We identify a metastable triple-helical conformation as the fundamental assembly unit, whose structural lability propagates through the assembly cascade, rendering the process sensitive to microenvironmental perturbations. Through hierarchical assembly, metastable intermediates achieve enhanced structural stability, culminating in the formation of stable collagen that retains its integrity under physiological conditions. In contrast, structural defects in intermediates lead to aberrant assembly and disruption of ECM integrity. Functional assays reveal that intermediates lacking D-band retain biological activity. Our findings redefine the fibrillar collagen assembly as a hierarchical, time-resolved cascade driven by metastable intermediates and propose the fundamental F-Z-F rules. The metastable triple helix provides a structural basis for hierarchical assembly and suggests a potential link between abnormal fibrillar collagen assembly and aging-related matrix dysfunction, offering valuable insights into collagen assembly and its role in aging diseases.

Objective: This study aims to systematically assess the therapeutic effects of Traditional Chinese Medicine (TCM) kidney-tonifying methods on osteoporosis in HR+ breast cancer patients after endocrine therapy, and to explore their clinical application value. Methods: This systematic review and meta-analysis adhered to the PRISMA guidelines and aimed to evaluate the impact of TCM kidney-tonifying methods on osteoporosis in HR+ breast cancer patients after endocrine therapy. Literature searches were conducted through October 2024 in databases including CBM, CNKI, Wanfang Data, PubMed, Web of Science, Cochrane Central, VIP, and renowned TCM databases such as TCM Online. Randomized controlled trials reporting lumbar spine bone density, femoral neck bone density, osteocalcin levels, pain VAS scores, and drug safety were included. The Cochrane tool was used for quality assessment, and meta-analysis was performed using RevMan 5.3 and Stata software. Results: This meta-analysis included 25 studies, covering 1795 patients. The combined results showed significant improvements in lumbar spine bone density (SMD = 0.98, 95 % CI: 0.52-1.44, P < 0.0001), femoral neck bone density (SMD = 0.73, 95 % CI: 0.35-1.10, P = 0.0001), and osteocalcin levels (SMD =1.23, 95 % CI: 0.52-1.93, P = 0.0007) in patients treated with TCM kidney-tonifying methods compared to those in the control group. There was also a significant reduction in pain VAS scores (SMD =-1.14, 95 % CI:1.55 to-0.72, P < 0.00001). The safety data indicated no significant adverse reactions associated with the treatment. Conclusion: TCM kidney-tonifying methods can significantly improve osteoporosis symptoms post- endocrine therapy in HR+ breast cancer patients, enhance bone density, and effectively reduce pain, demonstrating good safety and potential clinical value. Future research should further validate these findings through large-scale, multi-center randomized controlled trials to optimize treatment strategies.

The purpose of this study is to achieve the quantitative detection of recombinant type III collagen (rh-COL-III) in dressings with complex matrix. First of all, the marker peptide (GEAGIPGVPGAK) of rhCOL-III was identified with HPLC-MS/MS. Then, a qualitative and quantitative method based on marker peptides was established and validated. In order to obtain higher sensitivity, a pretreatment method of liquid, gel, and ointment dressings was optimized. The reference material for quantification was combined using rhCOL-III and blank matrix of each dressing. The results indicated that the relative standard deviation (RSD) of the quantitative method was 2.77%, and the RSD of intraday and interday precision was 2.76% and 2.31%, respectively. The spiking recovery rate was between 80% and 90%. The optimal pretreatment method was Tris-HCl solvent replacement. The optimal trypsin concentration for the dressing solution was 20 μg in 500 μL. The method of preparing standard substances with a blank matrix can effectively eliminate the influence of the matrix effect on the quantitative results. The average spiking recovery rates of 50 μg/mL, 100 μg/mL, and 200 μg/mL in three different dressings ranged from 80% to 120%. The quantitative detection of limit (LOD) of rhCOL-III was 1 ng/mL, 2 ng/g, and 1 ng/g in liquid, ointment, and gel dressings.

Animal tissue-derived decellularized extrocellular matrix（dECM） materials have similar matrix structure to human tissue. Due to their good biocompatibility and tissue inducing potential, they are widely used in the field of tissue repair and reconstruction clinically. The special risks caused by such materials are a series of unexpected material-mediated reactions, such as pyrogen, immunogen and inflammation caused by potential adventitious agents, xenogeneic cells and their immunogen residues and reagent residues in decellularization processes, and are needed to be paid attention to. Here, the risk analysis, risk control and risk evaluation of animal tissue-derived dECM materials were reviewed. Some relevant assessment technologies, methods and standards that have been preliminarily established at present were mainly reviewed and discussed. Finally, this paper looks forward to a more systematic quality evaluation system that needs to be improved.

Wild type (WT) animals cannot be used to objectively assess the immunogenicity of animal tissue-derived biomaterials when used as recipients due to difference with human in α-Gal expression. The purpose of this study is to compare the differences of immunological responses between the GGTA1 gene-knockout (GTKO) rabbits and WT rabbits after implantation with animal tissue-derived biomaterials. The porcine-derived decellularized bone matrix (natural bone material, NBM) and fresh porcine cancellous bone (PCB) were implanted in GTKO rabbits and WT rabbits, respectively, and sham operation was used as control (Con). At 2- and 6-week post-implantation, the related immunological items including antibody levels, serum-mediated cell lysis, cytokines, lymphocyte subtypes, and histopathological changes were assessed. GTKO rabbits exhibited more sensitive immune responses than WT rabbits after PCB implantation, resulted from a significant increase of antibodies (except total antibodies) and cytokines levels, cell lysis ratios, CD4/CD8 proportions, and inflammatory cells infiltration. Immunological factors and inflammatory cells infiltrate in GTKO rabbits after NBM implantation were significantly lower than those in the PCB group. Among the three groups, the NBM group showed the highest contents of new bone formation elements. In conclusion, the GTKO rabbit is a more sensitive alternative model than WT rabbit for preclinical study of xenografts via in situ implantation. Studies on multiple gene-edited animals are also necessary for more comprehensively evaluating xenoimmunologen risks of animal tissue-derived biomaterials in the future. Additionally, the immunogenicity of NBM was remarkably decreased compared to PCB.

对不同来源水产胶原的热稳定性差异以及水产胶原在生物医学领域应用的研究进展进行了综合性回顾,旨在为水产胶原原料的选择及应用提供有益借鉴.在水产胶原热稳定性能研究方面,重点综述不同鱼种、生长环境和组织部位对所得胶原热变性温度的影响及差异;在水产胶原材料的制备和应用研究方面,着重阐述水产胶原在创伤敷料、仿生骨材料、药物负载和缓释以及其他类型材料领域的应用进展.

Magnetic resonance imaging (MRI) is a promising non-invasive method to assess cartilage regeneration based on the quantitative relationship between MRI features and concentrations of the major components in the extracellular matrix (ECM). To this end, in vitro experiments are performed to investigate the relationship and reveal the underlying mechanism. A series of collagen (COL) and glycosaminoglycan (GAG) solutions at different concentrations are prepared, and T-1 and T-2 relaxation times are measured with or without a contrast agent (Gd-DTPA(2-)) by MRI. Fourier transform infrared spectrometry is also used to measure the contents of biomacromolecule-bound water and other water, allowing theoretical derivation of the relationship between biomacromolecules and the resulting T-2 values. It has been revealed that the MRI signal in the biomacromolecule aqueous systems is mainly influenced by the protons in hydrogens of biomacromolecule-bound water, which we divide into inner-bound water and outer-bound water. We have also found that COL results in higher sensitivity of bound water than GAG in T-2 mapping. Owing to the charge effect, GAG regulates the penetration of the contrast agent during dialysis and has a more significant effect on T-1 values than COL. Considering that COL and GAG are the most abundant biomacromolecules in the cartilage, this study is particularly useful for the real-time MRI-guided assessment of cartilage regeneration. A clinical case is reported as an in vivo demonstration, which is consistent with our in vitro results. The established quantitative relation plays a critical academic role in establishing an international standard ISO/TS24560-1:2022 'Clinical evaluation of regenerative knee articular cartilage using delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) and T-2 mapping' drafted by us and approved by International Standard Organization.

目的:建立可重复使用超声刀手术剪(简称超声刀)的清洗工艺确认方案,并对清洗有效性进行评价,为使用单位进行清洁方法确认和确保安全使用提供依据.方法:选择模拟血液污染物,按照确定的清洗工艺,选择最严苛的清洁方法挑战条件,通过对超声刀的可复用部件刀杆进行污染物负载-清洗数次后,提取残留污染物,并检测残留污染物中总蛋白和血红蛋白残留量.根据美国食品药品监督管理局(FDA)相关指导原则、美国医疗器械促进协会(AAMI)及美国材料和试验协会(ASTM)相关标准,设定可复用超声刀满足清洁要求的残留物限量指标(总蛋白残留量<6.4μg/cm2,血红蛋白残留量<2.2μg/cm2).结果:所建立的确认方案体系中,残留污染物的提取回收率均>79％;彻底提取验证研究中,单次提取回收率达到4次彻底提取回收的90％以上,满足实验要求.负载-清洗1次、4次、6次和8次清洗后,可重复使用超声刀总蛋白残留量均未检出,低于方法检测限(0.43μg/cm2);血红蛋白残留量均<0.34μg/cm2,低于确认方案体系的残留污染物中血红蛋白检测下限(1.10μg/cm2).结论:本研究建立的可复用超声刀手术剪清洁方法确认方案及清洁有效性评价方法;经验证,可重复使用超声刀8次,污染物负载-清洗后仍能满足所规定的清洁要求.

The purpose of this study was to establish a collagen determination method based on an isotope-labeled collagen peptide as an internal reference via high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS), and using the established method to evaluate the degradation process of collagen-based implants in vivo. The specific peptide (GPAGPQGPR) of bovine type I collagen was identified with an Orbitrap mass spectrometer. Then, the quantification method based on the peptide detection with HPLC-MS/MS was established and validated, and then further used to analyze the degradation trend of the collagen sponge and acellular matrix (ACM) in vivo at 2, 4, 6, 8, 12, 16, and 18 weeks after implantation. The results indicate that the relative standard deviation (RSD) of the detection precision and repeatability of the peptide-based HPLC-MS/MS quantification method were 3.55% and 0.63%, respectively. The limitations of quantification and detection were 2.05 × 10−3 μg/mL and 1.12 × 10−3 μg/mL, respectively. The collagen sponge and ACM were completely degraded at 10 weeks and 18 weeks, respectively. Conclusion: A specific peptide (GPAGPQGPR) of bovine type I collagen was identified with an Orbitrap mass spectrometer, and a standardized HPLC-MS/MS-based internal reference method for the quantification of bovine type I collagen was established. The method can be used for the analysis of the degradation of collagen-based implants in vivo.

Peripheral nerve injury (PNI) is a neurological disorder caused by trauma that is frequently induced by accidents, war, and surgical complications, which is of global significance. The severity of the injury determines the potential for lifelong disability in patients. Artificial nerve scaffolds have been investigated as a powerful tool for promoting optimal regeneration of nerve defects. Over the past few decades, bionic scaffolds have been successfully developed to provide guidance and biological cues to facilitate Schwann cell myelination and orientated axonal growth. Numerous assessment techniques have been employed to investigate the therapeutic efficacy of nerve scaffolds in promoting the growth of Schwann cells and axons upon the bioactivities of distinct scaffolds, which have encouraged a greater understanding of the biological mechanisms involved in peripheral nerve development and regeneration. However, it is still difficult to compare the results from different labs due to the diversity of protocols and the availability of innovative technologies when evaluating the effectiveness of novel artificial scaffolds. Meanwhile, due to the complicated process of peripheral nerve regeneration, several evaluation methods are usually combined in studies on peripheral nerve repair. Herein, we have provided an overview of the evaluation methods used to study the outcomes of scaffold-based therapies for PNI in experimental animal models and especially focus on Schwann cell functions and axonal growth within the regenerated nerve.

Recombinant collagen, as an alternative to natural collagen, has the potential to be widely used in biomaterials, biomedicine, etc. Diverse recombinant collagens and their variants can be industrially produced in a variety of expression systems, which lays a foundation for exploring and expanding the clinical application of recombinant collagens. We reviewed different expression systems for recombinant collagens, such as prokaryotic expression systems, yeast expression systems, as well as plant, insect, mammal, and human cell expression systems, and introduced the advantages, potential applications, and limitations of recombinant collagen. In particularly, we focused on the current progress in the recombinant collagen production, including recombinant expression system construction and hydroxylation strategies of recombinant collagen, and summarized the current biomedical applications of recombinant collagen.

Efficient and large-scale expansion of mesenchymal stem/stromal cells (MSCs) has always been a formidable challenge to researchers in cell-based therapies and regenerative medicine. To reconcile major drawbacks of 2D planar culturing system, we innovatively developed an automated closed industrial scale cell production (ACISCP) platform based on GMP-grade microcarrier for culture of umbilical cord-mesenchymal stem/stromal cells (UCMSCs), in accordance with the criteria of stem cell bank. ACISCP system is a fully closed system, which employs different models of vivaSPIN bioreactors (CytoNiche Biotech, China) for scale-up cell culture and vivaPREP (CytoNiche Biotech, China) for automated cell harvesting and cell dosage preparation. To realize industrial scale expansion of UCMSCs, a three-stage expansion was conducted with 1 L, 5 and 15 L vivaSPIN bioreactors. Using 3D TableTrix® and ACISCP system, we inoculated 1.5 × 107 of UCMSCs into 1 L vivaSPIN bioreactor and finally scaled to two 15 L bioreactor. A final yield of 2.09 × 1010 cells with an overall expansion factor of 1975 within 13 days. The cells were harvested, concentrated, washed and prepared automatically with vivaPREP. The entire process was realized with ACISCP platform and was totally enclosed. Critical quality attributes (CQA) assessments and release tests of MSCs, including sterility, safety, purity, viability, identity, stability and potency were performed accordingly. The quality of cells harvested from 3D culture on the ACISCP and conventional 2D planar culture counterpart has no significant difference. This study provides a bioprocess engineering platform, harnessing GMP-grade 3D TableTrix® microcarriers and ACISCP to achieve industrial-scale manufacturing of clinical-grade hMSCs.

Abstract Background Previous studies have identified the carbohydrate epitope Galα1–3Galβ1–4GlcNAc-R (termed the α-galactosyl epitope), known as the α-Gal antigen as the primary xenoantigen recognized by the human immune system. The α-Gal antigen is regulated by galactosyltransferase (GGTA1), and α-Gal antigen-deficient mice have been widely used in xenoimmunological studies, as well as for the immunogenic risk evaluation of animal-derived medical devices. The objective of this study was to develop α-Gal antigen-deficient rabbits by GGTA1 gene editing with the CRISPR/Cas9 system. Results The mutation efficiency of GGTA1 gene-editing in rabbits was as high as 92.3% in F0 pups. Phenotype analysis showed that the α-Gal antigen expression in the major organs of F0 rabbits was decreased by more than 99.96% compared with that in wild-type (WT) rabbits, and the specific anti-Gal IgG and IgM antibody levels in F1 rabbits increased with increasing age, peaking at approximately 5 or 6 months. Further study showed that GGTA1 gene expression in F2-edited rabbits was dramatically reduced compared to that in WT rabbits. Conclusions α-Gal antigen-deficient rabbits were successfully generated by GGTA1 gene editing via the CRISPR/Cas9 system in this study. The feasibility of using these α-Gal antigen-deficient rabbits for the in situ implantation and residual immunogenic risk evaluation of animal tissue-derived medical devices was also preliminarily confirmed.

目的:采用CFSE法与CCK-8法评价同种异体骨对人外周血淋巴细胞的活性与增殖效应.方法:通过人新鲜外周血淋巴细胞与供试品(脱钙骨基质与冻干型组织工程骨)浸提液共培养后,采用CCK-8法检测淋巴细胞的活性水平.通过CFSE标记的人新鲜外周血淋巴细胞与供试品浸提液共培养后,采用流式细胞术检测淋巴细胞增殖能力.结果:CCK-8法试验结果表明,脱钙骨基质组的吸光度值与培养液对照组相比无显著性差异,而冻干型组织工程骨组的吸光度值与培养液对照组相比显著增高(P＜0.05),表明冻干型组织工程骨提高了淋巴细胞的活性.CFSE法试验结果表明,与培养液对照组相比,冻干型组织工程骨组的淋巴细胞增殖相关指标(Dil、PF、EI、RI)以及淋巴细胞亚群增殖指标Dil均无显著性差异(P＞0.05),表明冻干型组织工程骨未对人外周血淋巴细胞的增殖产生显著性影响.结论:与培养液对照组相比,冻干型组织工程骨提高了人外周血淋巴细胞的活性,但是未对人外周血淋巴细胞的增殖产生显著影响.CCK-8法主要反映的是细胞活性,而CFSE法可以更客观地反映细胞有无增殖.

OBJECTIVE:In this study, α-Gal epitope-deficient (GGTA1 knockout (GTKO)) mice were used to assess the immunological risks of xenogeneic dural patch by comparing with raw material.METHODS:The xenogeneic dural patch (T2) was prepared from bovine pericardium (T1, raw material) through decellularization and carboxymethyl chitosan (CMCS) coating. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were used to characterize the collagen fibers and surface microstructural changes in the T1 and T2 samples. The remnant α-Gal epitopes and DNA of implants were detected by standardized method. T1 and T2 were implanted subcutaneously into GTKO mice for 4 and 12 weeks, respectively, and the negative control group (Con) was only performed sham operation. The total serum antibody, anti-Gal antibody, and splenic lymphocyte subtypes were analyzed by ELISA or flow cytometry, and histological analysis of implant-tissue was performed by H&E and Masson stain.RESULTS:TEM and Sirius red staining showed that the collagen fibers in the dural patch were closely arranged, and SEM showed that a loose three-dimensional structure was successfully constructed on the surface of the dural patch after CMCS coating. The remnant DNA in T2 was 24.64 ± 8.73 ng/mg (dry weight), and clearance of α-Gal epitope was up to 99.83% compared to T1. The significant increases in serum total IgM, anti-Gal IgG, and anti-Gal IgM at 4 weeks and the significant changes in anti-Gal IgG and spleen lymphocyte at 12 weeks were observed in the T1 group, but no significant change was observed in the T2 group, compared to the control group. Histological semiquantitative analysis showed severe cell and tissue responses at 4 weeks and a moderate response at 12 weeks in the T1 group, while a moderate response at 4 weeks and a slight response at 12 weeks in the T2 group.CONCLUSIONS:The results demonstrated that the xenogeneic dural patch has a lower and acceptable immunological risk compared to the raw material and control, respectively. On the other hand, it was suggested that GTKO mice are useful experimental model for immunological risk assessment of animal tissue-derived biomaterials.

目的:采用腹腔巨噬细胞吞噬鸡红细胞试验考察Gal抗原缺失小鼠评价动物源材料对固有免疫影响的敏感性.方法:Gal抗原缺失小鼠(GGTA1 KO)经兔血预免后,随机分为空白对照组(生理盐水)、地塞米松抑制性阳性对照组、植物血凝素刺激性阳性对照组、牛跟腱样品植入组.地塞米松阳性对照组与植物血凝素阳性对照组参照YY/T 1465.4-2017进行给药.牛跟腱植入组皮下植入牛跟腱(2.4 cm2/只),周期为4周.最后所有实验组小鼠均腹腔注射鸡血红细胞,参照YY/T 1465.4-2017计数巨噬细胞和被吞噬的鸡血红细胞个数,并计算吞噬百分数和吞噬指数.结果:Gal抗原缺失小鼠的植物血凝素阳性对照组的细胞吞噬百分数和吞噬指数为39.05％和2.02,略高于空白对照组的32.15％和1.87,但无统计学差异;牛跟腱植入组的细胞吞噬百分数和吞噬指数分别为55％和2.59,均高于空白对照组,存在显著性差异(P＜0.01);地塞米松抑制性阳性对照组吞噬百分数和吞噬指数分别为20.60％和1.43,低于空白对照组(P＜0.05,P＜0.01);研究结果表明,Gal抗原缺失小鼠对植物血凝素有一定程度的刺激反应,而地塞米松可明显降低吞噬百分数和吞噬指数,说明试验体系基本成立.牛跟腱植入物引起了吞噬细胞吞噬百分数和吞噬指数的显著升高,提示牛跟腱具有免疫原性风险.结论:在腹腔巨噬细胞吞噬鸡血红细胞试验中,Gal抗原缺失小鼠对地塞米松抑制性阳性对照表现出显著的抑制效应,而牛跟腱植入物的刺激引起了明显的刺激效应,表明Gal抗原缺失小鼠对于评价动物源性生物材料对固有免疫的影响具有一定应用价值.

With the development of tissue engineering and regenerative medicine, it is much desired to establish bioimaging techniques to monitor the real-time regeneration efficacy in vivo in a non-invasive way. Herein, we tried magnetic resonance imaging (MRI) to evaluate knee cartilage regeneration after implanting a biomaterial scaffold seeded with chondrocytes, namely, matrix-induced autologous chondrocyte implantation (MACI). After summary of the T2 mapping and the T1-related delayed gadolinium-enhanced MRI imaging of cartilage (dGEMRIC) in vitro and in vivo in the literature, these two MRI techniques were tried clinically. In this study, 18 patients were followed up for 1year. It was found that there was a significant difference between the regeneration site and the neighboring normal site (control), and the difference gradually diminished with regeneration time up to 1 year according to both the quantitative T1 and T2 MRI methods. We further established the correlation between the quantitative evaluation of MRI and the clinical Lysholm scores for the first time. Hence, the MRI technique was confirmed to be a feasible semi-quantitative yet non-invasive way to evaluate the in vivo regeneration of knee articular cartilage.

目的:研究乙醇工艺对同种异体肌腱的病毒灭活效果.方法:选择伪狂犬病毒(PRV)、猪细小病毒(PPV)、牛腹病毒性腹泻病毒(BVDV)和人类免疫缺陷病毒(HIV-1)为指示病毒,将同种异体肌腱(3个批号)分别负载指示病毒后,用75％乙醇浸泡灭活处理;分别在不同时间点取样,用96孔板的细胞病变法和Karber法测定,计算乙醇工艺处理后同种异体肌腱的病毒残留量(LgTCID50/0.1mL)和计算病毒降低量(log).结果:负载病毒的3个批号样品经过75％乙醇浸泡2h后,PRV、BVDV和HIV-1滴度降低量分别为≥5.971 logs、≥4.400 logs和≥8.000 logs;病毒灭活动力曲线显示:病毒残留量很快降到最低检出限度值.负载PPV的3个批号样品,经75％乙醇浸泡2h后,病毒滴度降低量平均为0.850 logs,浸泡延长至24 h后,病毒滴度降低量平均为1.350 logs,病毒灭活动力曲线显示病毒滴度随时间呈缓慢降低趋势.结论:75％乙醇工艺浸泡处理同种异体肌腱2h,对PRV、BVDV和HIV-1为有效工艺,对PPV无效.

背景:有研究表明一种称为α-Gal的糖抗原是动物源性生物材料或异种器官移植引起超急性免疫排斥反应的主要靶抗原.目的:研制Gal抗原缺失兔模型,预期用于评价植入性医疗器械,其局部植入后对宿主的局部免疫反应及非特异性炎症反应风险.方法:选用SPF级6-8月龄新西兰大白兔作为模式动物蓝本,制备Gal抗原缺失兔模型.采用CRISPR/Cas9介导的基因编辑技术,针对调控兔子Gal抗原表达的GGTA1基因第8外显子设计构建2条相辅的sgRNA.经转录后将GGTA1 sgRNA mRNAs与Cas9 mRNA共显微注射到体外培养的兔子受精卵中,继续短暂体外培养后植入代孕母兔体内,经自然妊娠获得仔兔.基因编辑成功与否通过凝胶电泳和基因测序进行验证.Gal抗原的表达参照行业标准给出的方法(YY/T 1561-2017)进行检测.研究经中国食品药品检定研究院实验动物资源研究所动物伦理委员会批准[批准号:中检动(福)第2017(B)007号].结果与结论:①基因编辑后的胚胎被转移至4只代孕兔体内,自然妊娠后获得15只基因编辑仔兔,仔兔的外观发育及进食行为等未见异常;②凝胶电泳及基因测序结果显示15只仔兔中有14只的靶向基因被成功编辑,但编辑的碱基并不相同;随机检测其主要脏器Gal抗原,表达均降低了99.96％以上;③结果表明,Gal抗原缺失兔子有望用于动物源性医疗器械的免疫原性风险评价和异种骨、角膜等原位植入实验,以便能够更客观科学地评价动物源性医疗器械的安全性和有效性.

目的:建立猪细小病毒(PPV)的细胞培养结合荧光定量PCR( ICC-qPCR)方法并进行优化,结合传统的病毒滴定(细胞培养法)及ICC-qPCR方法,考察将其应用于病毒灭活验证研究的可行性.方法:将系列10倍稀释的PPV病毒接种于猪睾丸(ST)细胞,分别扩增培养0、12、18、24 h,考察理想的病毒扩增培养时间,确定ICC-qPCR定量检测区间及获得病毒扩增倍数(K);将病毒平行接种于病毒扩增组及非扩增的对照组,通过扩增组和对照组的系列病毒接种量与PCR反应周期(Ct)值的拟合曲线,分别获得病毒扩增组及对照组的ICC-qPCR检测病毒滴度(Ta、Tc),结合扩增倍数(K),根据公式[Log10( l0Ta-10fc)/(K-l)]直接计算样本中的感染性病毒滴度.最后,将ICC-qPCR、优化ICC-qPCR方法和细胞培养法分别用于模拟样本(由不同比例感染性病毒和非感染性病毒组成)中的感染性病毒滴度测定,考察将其应用于病毒灭活验证研究的可行性.结果:PPV接种后的最佳扩增培养时间为24 h,检测下限为-1 Log10TCID50 · 100μL-1(Logs),定量区间为0～5.00 Logs,扩增倍数为83.11.当模拟样本中的非感染性病毒含量较低或与感染性病毒等量时,ICC-qPCR、优化ICC-qPCR方法和细胞培养法测得的感染性病毒滴度没有显著性差异;然而,当模拟样本中的非感染性病毒含量较高时,由优化的ICC-qPCR方法和细胞培养法测得的感染性病毒滴度分别是1.46、1.50 Logs,二者高度一致,而常规ICC-qPCR方法测得的结果(1.75 Logs )存在极显著性差异.结论:本研究所优化的ICC-qPCR方法作为细胞培养法的替代方法,具有用于病毒灭活验证研究的前景.