Establishment of a Spleen Cell Line from Large Yellow Croaker Pseudosciaena crocea and its Primitive Application in Foreign Gene Transfection

A large yellow croaker,Pseudosciaena crocea,spleen(LYCS)cell line was established and the feasibility of using it for foreign gene transfection was evaluaed in this study.Primary culture of LYCS cells was initiated from spleen tissue pieces,which were cultured at 25℃ in Dulbecco's modiced Eagle medium/F12 medium(DMEM/F12,1:1)(pH7.2),supplemented with 20% fetal bovine serum,carboxymethyl chitosan,chondroitin sulfate,basic fibroblast growth factor(bFGF)and insulin-like growth factor-I(IGF-I).The cultured LYCS cells,in fibroblast shape,proliferated to 100% confluency 20 days later.Chromosome analyses indicated that the LYCS cells exhibited chromosomal aneuploidy with a modal chromosome number of 48 which displayed the normal diploid karyotype of P.crocea(6m+6sm+36t,NF=60).A LYCS cell line,with a population doubling time of 48.7 h at passage 60,has been established and subcultured to passage 70.Transgenic feasibility test demonstrated that positive green fluorescence protein(GFP)expression was observed in LYCS cells after pcDNA3.1-GFP plasmid transfection.In conclusion,a continuous foreign gene trans-fection feasible LYCS cell line has been established successfully.The cell line might serve as a valuable tool for studies of transgenic breeding and has potential applications for different kinds of cytotechnological studies.

Preoperative application of combination of portal venous injection of donor spleen cells and intraperitoneal injection of rapamycin prolongs the survival of cardiac allografts in mice

Objective:To investigate the effects of preoperative portal venous injection of donor spleen cells(PVIDSC) and intraperitoneal injection of rapamycin in the acute rejection of cardiac allograft in mice and the underlying mechanisms. Methods:Homogenous female B6 mice and BALB/c mice were used as recipients and donors of heart transplantation. These mice were randomly divided into different groups and received PVIDSC alone,rapamycin alone,or PVIDSC and rapamycin combined therapy. In addition,the underlying mechanism was studied by measuring a number of cytokines. Results:Preoperative combination of PVIDSC and intraperitoneal injection of rapamycin significantly prolonged the survival of heterotopic cardiac allograft in mice,but had no effects on the survival time of cardiac allografts in mice pre-sensitized by skin grafting. Preoperative combination of PVIDSC and intraperitoneal injection of rapamycin increased the expression of IL-10 and Foxp3 and reduced the expression of INF-γ. Short-term preoperative administration of rapamycin promotes the expression of CD4^+CD25^+Foxp3^+ regulator T cells. However,preoperative using alone of rapamycin,or combination of PVIDSC and rapamycin had no effects on the inhibition of proliferation of memory T cells. Conclusions:Preoperative application of combination of PVIDSC and rapamycin significantly prolonged the survival time of cardiac allografts in mice but not in mice pre-sensitized by skin grafting. This may be explained by the fact that combination of PVIDSC and rapamycin inhibited the cellular immune response and induced the expression of IL-10 from Tr1 cells and CD4^+CD25^+FoxP3^+ regulatory T cells.

Littoral-cell angioma of the spleen:a case report

Littoral-cell angioma(LCA), a primary angioma which clinically belongs to splenic hemangioma, can be mostly found in normal spleen red sinus shore cells of reticuloendothelial cell system. The cells of LCA strongly express endothelial and tissue cell associated antigens that indicate a dual differentiation characteristic; whereas only endothelial cell markers are positive in normal spleen red sinus shore cells. Diagnosis of LCA relies on histopathology. Regular follow-up is needed to monitor recurrence and metastasis.

Functional distinction of rat liver natural killer cells from spleen natural killer cells under normal and acidic conditions in vitro

BACKGROUND: The microenvironment within solid tumors has often been shown to exhibit an acidic extracellular pH. Although the morphologic and functional differences in natural killer (NK) cells of the liver and spleen have been reported previously under physiological conditions, the difference under acidic conditions is still unclear. This study was to investigate the differences in the morphological and functional characteristics between rat liver and spleen NK cells under normal and acidic conditions in vitro. METHODS: Liver and spleen NK cells were isolated and purified from Sprague-Dawley rats by density gradient centrifugation and the Dynabeads FlowComp TM Flexi system, and stimulated for 4 days with or without IL-2 or treated with low pH or control for different times. Morphology was examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), cell death and proliferation assays were performed by flow cytometry, IFN-γ production was tested by ELISA, and cytotoxic activity was evaluated by lactate dehydrogenase (LDH) release assay. RESULTS: Liver NK cells had significantly higher levels of cytotoxic activity than spleen NK cells under normal and acidic conditions, and the maximum difference was observed at pH 5.6. Further analysis revealed that the cytotoxic activity of NK cells was correlated with morphology, cell death, proliferative activity and IFN-γ production. By TEM, liver NK cells contained a greater number of electron-dense granules per cell at pH 5.6.Moreover, a modest elevation of cell death and reduction of proliferation of liver NK cells occurred within a range of 5.6-7.2. Interestingly, an acidic extracellular pH only marginally, and not significantly, suppressed IFN-γ production by liver NK cells. CONCLUSION: The sharp morphological and functional differences shown by the two types of NK cells in vitro indicate that liver NK cells are unexpectedly resistant to pH shock.

Littoral cell angiomas of the spleen associated with solid pseudopapillary tumor of the pancreas

Littoral cell angiomas(LCA) of the spleen are vascular tumors of unknown etiology arising from the littoral cells of the splenic red pulp sinuses. Usually a benign and incidental finding,LCA have been repeatedly reported in association with a variety of visceral malignancies and hold the potential for dissemination per se. We encountered a case of a 30 year old female who was diagnosed with solid pseudopapillary tumor of the head and distal pancreas by fine needle aspiration cytology. A distal pancreatectomy with splenectomy was performed in addition to a pylorus-preserving Whipple's procedure and cholecystectomy. Histopathological examination confirmed solid pseudopapillary tumor of the pancreasand showed multiple well-circumscribed anastomosing vascular channels in the spleen. The diagnosis of LCA of the spleen was confirmed by immunohistochemistry that revealed co-expression of endothelial cell marker,CD31 and CD34,along with histiocytic marker,CD68 by the vascular lining cells. LCA has been previously reported in association with colorectal and pancreatic adenocarcinoma,malignant lymphoma,myelodysplasia and autoimmune disorders. We report the first case of LCA associated with solid pseudopapillary tumor of the pancreas.

The Spleen and Sickle Cell Anemia: A Contrast Enhanced Computerized Tomography Based Study

The spleen is one of the most frequently affected organs in sickle cell anemia (SCA). This study aims to characterize the spleen in sickle cell anemia patients using contrast enhanced computerized tomography scanning (CECT). 67 patients with SCA from different Saudi Arabian areas were enrolled;ages are ranged from 10 months to 28 years old. The spleen was assessed with CT for abdominal pain and/or unexplained fever. The evaluation was done at different contrast enhancement scanning phases. The study showed that the least number of affected patients was from Eastern Saudi Arabia (1.5%) followed by Asseer (16.4%) then Gazan representing 82.1%. The most common type of SCA affected the Saudi children is Hemoglobin SS Disease (Hb SS) constituting 41 (61.2%). The spleen size, lymph nodes size, spleen Hounsfield (HU), splenic vein diameter and the correlation with the associated findings were evaluated for all of the patients. In children affected with SCA: 26 (38.8%) have splenomegaly, 18 (26.9%) have atrophied spleen and 2 (3.0%) are with very small tissue like structure. Lesions found in the spleen were abscess, infarction, cyst, and calcifications. At the spleen hilum region;dilated splenic vein, presence of multiple collaterals, and thrombus were also been detected. Significantly correlations were noticed between lesions type, child age, enlargement of spleen and splenic lymph nodes at P ≤ 0.033, P ≤ 0.010 and P ≤ 0.012 respectively and showed an evidence that the reduction of the HU and advanced age have significant relation with changing of the spleen size at P ≤ 0.004 and P ≤ 0.000 respectively. Spleen lesions’ enhancement pattern is well emerged in both venous and delay phase and it was significantly related with the scanning phase at P ≤ 0.000 and with different types of SCA at P ≤ 0.037, and P ≤ 0.055 in venous and delay phase in respectively. CECT offers a number of morphological criteria that can be applied to differentiate hypodense lesions of the spleen in SCA. CT characterization criteria of hypodense splenic lesions are acknowledged to aid interpretation during evaluation of abdominal CT images of the spleen in symptomatic patients with sickle cell anemia.

REGULATORY EFFECTS OF HUMAN RECOMBINANT IL-6 ON NATURAL KILLER CELL ACTIVITY OF HUMAN FETAL SPLEENS

In the present study it was proved first that human recombinant interleukin-6(HrIL-6) significantly augmented natural killer(NK) cell activity derived from human fetal spleens against K562 target cells in a 4 hours 51Cr release assay. The enhancement of NK activity with 24 hours preincubation in HrlL-6 was dose-dependent, and significantly higher than that of fresh NK cells and controls cultured with RPMI-1640 medium alone (P<0.001). We also found that IL-6 was able to augment NK activity from different fetal spleens at 20 to 40 weeks of gestation (up to 2.24 to 2.78 times), and no difference of NK activity of fetal splenocytes treated by HrIL-6 was observed between different fetal age (32.3% to 45.4%, P>0.05). Furthermore, IL-6-augmented NK activity of fetal splenocytes was very similar to adult levels (P>0.05). These finding strongly indicated that IL-6 plays an important role in the development of NK cell function during the gestational period, suggesting that IL-6 may be of importance in the regulation of host defense mechanisms against malignancies and viral diseases.

EFFECTS OF MEDICINAL MOXIBUSTION ON SERUM GASTRIN,URINARY D-XYLOSE EXCRETORY RATE AND T CELL SUBGROUPS IN SPLEEN DEFICIENCY PATIENTS

The medicinal moxa stick made by moxa wool combining with Chinese medicines of supporting the body resistance and strengthening the spleen was used to treat 74 cases of spleen deficiency, the other 41 patients as control were treated by pure moxa stick. After treatment, the serum gastrin, urinary D - xylose excretory rate and T cell subgroups in patients with spleen deficiency have been greatly improved as compared with those before treatment in two groups (P< 0.01 or 0.05). Comparing the changes of the indexes between treatment group and control group, there were significant(P<0.05), or markedly significant(P<0.01) differences. It indicated that the medicinal moxibustion could improve the secreting and absorbing functions of digestive tract and the cellular immune function in patients with spleen deficiency.

高活性脐带间充质干细胞干预老年树鼩衰老脾脏的作用与机制

背景:脾脏具有储血、造血和免疫功能,随着年龄增长,脾脏结构退变、功能衰退引起免疫系统功能受损,进而加速机体衰老进程,而高活性脐带间充质干细胞治疗树鼩脾脏衰老尚未见报道。目的:探讨高活性脐带间充质干细胞对树鼩脾脏衰老的干预作用及机制。方法:从剖腹产的新生树鼩脐带组织中分离、培养和获得高活性脐带间充质干细胞,三系分化试剂盒检测成脂、成骨、成软骨分化能力,流式细胞术检测细胞周期和表面标志物。以感染复数值分别为100,120,140,160,180,200的吉凯基因绿色荧光蛋白转染第2代高活性脐带间充质干细胞,筛选最佳转染条件;转染后的第4代高活性脐带间充质干细胞尾静脉输注给老年治疗组树鼩,青年对照组和老年模型组不予特殊处理,治疗4个月时取脾脏组织,苏木精-伊红染色观察脾脏组织结构;β-半乳糖苷酶染色检测衰老相关半乳糖苷酶活性;免疫组织化学染色检测p21和p53蛋白表达水平;Ki67和PCNA免疫荧光染色检测细胞增殖活性;免疫荧光染色检测脾脏自噬蛋白分子Beclin1和APG5L/ATG5表达水平;活性氧荧光染色检测脾脏组织的活性氧含量;CD3免疫荧光染色检测总T淋巴细胞比例变化;酶联免疫吸附实验检测脾脏组织白细胞介素1β和转化生长因子β1分泌水平;DAPI复染细胞核观察绿色荧光蛋白标记的高活性脐带间充质干细胞在脾脏组织的分布情况。结果与结论:①高活性脐带间充质干细胞呈核小短梭形、鱼群样生长,G_(0)/G_(1)期占比大,具有向成脂、成骨和成软骨分化潜能。②感染复数为140且转染72 h为吉凯基因绿色荧光蛋白标记树鼩高活性脐带间充质干细胞的最佳条件。③与老年模型组相比,老年治疗组树鼩脾脏组织细胞排列紧密,白髓面积增加(P<0.01),红白髓界线清晰,生发中心占比无显著差异(P>0.05),脾脏组织衰老相关半乳糖苷酶活性水平降低(P<0.001),衰老蛋白分子p21和p53表达下调(P<0.001),增殖相关分子Ki67和PCNA表达上调(P<0.001,P<0.05),自噬相关分子Beclin1和APG5L/ATG5表达上调(P<0.001),活性氧含量降低(P<0.001),CD3^(+)T细胞比例增加(P<0.05),衰老相关分泌表型中白细胞介素1β分泌水平降低(P<0.001),转化生长因子β1分泌水平无显著差异(P>0.05)。与青年对照组相比,以上检测指标在老年治疗组中均有显著差异(P<0.05)。④冰冻组织切片观察可见老年治疗组树鼩脾脏组织中有绿色荧光蛋白标记的绿色荧光细胞。结果表明:静脉输注高活性脐带间充质干细胞可迁移至脾脏组织,抑制活性氧产生,下调衰老相关蛋白分子表达,诱导细胞自噬,促进细胞增殖,降低慢性炎症,进而改善脾脏组织结构和功能。

Effect of a cancer vaccine prepared by fusions of hepatocarcinoma cells with dendritic cells

AIM: To prepare a cancer vaccine (H(22)-DC) expressing high levels of costimulatory molecules based on fusions of hepatocarcinoma cells (H(22)) with dendritic cells (DC) of mice and to analyze the biological characteristics and induction of specific CTL activity of H(22)-DC. METHODS: DCs were isolated from murine spleen by metrizamide density gradient centrifugation, purified based on its characteristics of semi-adhesion to culture plates and FcR-,and were cultured in the medium containing GM-CSF and IL-4. A large number of DC were harvested. DCs were then fused with H(22) cells by PEG and the fusion cells were marked with CD11c MicroBeads. The H(22)-DC was sorted with Mimi MACS sorter. The techniques of cell culture, immunocytochemistry and light microscopy were also used to test the characteristics of growth and morphology of H(22)-DC in vitro. As the immunogen, H(22)-DC was inoculated subcutaneously into the right armpit of BALB/C mice, and their tumorigenicity in vivo was observed. MTT was used to test the CTL activity of murine spleen in vivo. RESULTS: DC cells isolated and generated were CD11c+ cells with irregular shape, and highly expressed CD80, CD86 and CD54 molecules. H22 cells were CD11c- cells with spherical shape and bigger volume, and did not express CD80, CD86 and CD54 molecules.H(22)-DC was CD11c+ cells with bigger volume, being spherical, flat or irregular in shape, and highly expressed CD80, CD86 and CD54 molecules, too. H(22)-DC was able to divide and proliferate in vitro, but its activity of proliferation was significantly decreased as compared with H(22) cells and its growth curve was flatter than H(22) cells. After subcutaneous inoculation over 60 days, H(22)-DC showed no tumorigenecity in mice, which was significantly different from control groups (P【0.01). The spleen CTL activity against H(22) cells in mice implanted with fresh H(22)-DC was significantly higher than control groups (P 【 0.01). CONCLUSION: H(22)-DC could significantly stimulate the specific CTL activity of murine spleen, which suggests that the fusion cells have already obtained the function of antigen presenting of parental DC and could present H(22)specific antigen which has not been identified yet, and H(22)-DC could induce antitumor immune response; although simply mixed H(22) cells with DC could stimulate the specific CTL activity which could inhibit the growth of tumor in some degree, it could not prevent the generation of tumor. It shows that the DC vaccine is likely to become a helpful approach in immunotherapy of hepatocarcinoma.

Liver regeneration using decellularized splenic scaffold: a novel approach in tissue engineering

BACKGROUND： The potential application of decellularized liver scaffold for liver regeneration is limited by severe shortage of donor organs. Attempt of using heterograft scaffold is accompanied with high risks of zoonosis and immunological rejection. We proposed that the spleen, which procured more extensively than the liver, could be an ideal source of decellularized scaffold for liver regeneration. METHODS： After harvested from donor rat, the spleen was processed by 12-hour freezing/thawing ×2 cycles, then circulation perfusion of 0.02% trypsin and 3% Triton X-100 sequentially through the splenic artery for 32 hours in total to prepare decellularized scaffold. The structure and component characteristics of the scaffold were determined by hematoxylin and eosin and immumohistochemical staining, scanning electron microscope, DNA detection, porosity measurement, biocompatibility and cytocompatibility test. Recellularization of scaffold by 5×106 bone marrow mesenchymal stem cells（BMSCs） was carried out to preliminarily evaluate the feasibility of liver regeneration by BMSCs reseeding and differentiation in decellularized splenic scaffold.RESULTS： After decellularization, a translucent scaffold, which retained the gross shape of the spleen, was generated. Histological evaluation and residual DNA quantitation revealed the remaining of extracellular matrix without nucleus and cytoplasm residue. Immunohistochemical study proved the existence of collagens I, IV, fibronectin, laminin and elastin in decellularized splenic scaffold, which showed a similarity with decellularized liver. A scanning electron microscope presented the remaining three-dimensional porous structure of extracellular matrix and small blood vessels. The poros-ity of scaffold, aperture of 45.36±4.87 μm and pore rate of 80.14%±2.99% was suitable for cell engraftment. Subcutaneous implantation of decellularized scaffold presented good histocompatibility, and recellularization of the splenic scaffold demonstrated that BMSCs could locate and survive in the decellularized matrix. CONCLUSION： Considering the more extensive organ source and satisfying biocompatibility, the present study indicated that the three-dimensional decellularized splenic scaffold might have considerable potential for liver regeneration when combined with BMSCs reseeding and differentiation.

Inflammatory pseudotumor-like follicular dendritic cell sarcoma: A brief report of two cases

BACKGROUND Follicular dendritic cell(FDC)sarcoma/tumor is a rare malignant tumor of follicular dendritic cells,which is considered a low-grade sarcoma that can involve lymph nodes or extranodal sites.Conventional FDC sarcomas are negative for Epstein-Barr virus(EBV),whereas the inflammatory pseudotumorlike variant consistently shows EBV in the neoplastic cells.CASE SUMMARY We report two cases of inflammatory pseudotumor-like FDC sarcoma in the liver that received 3D laparoscopic right hepatectomy and open right hepatectomy separately.CONCLUSION EBV probe-based in situ hybridization and detection of immunohistochemical markers of FDC play an important role in the diagnosis and differential diagnosis of inflammatory pseudotumor-like FDC sarcoma.Complete surgical excision combined with regional lymphadenectomy may be effective in reducing the postoperative recurrence and metastasis and improving long-term survival rates.

“脾肾相赞”理论探讨不同治法对大鼠骨髓间充质干细胞成肌分化中TNF-α、JNK含量影响

目的在“脾肾相赞”理论指导下,比较不同中医治法对大鼠BMSCs成肌分化及过程中TNF-α、JNK蛋白表达的影响,分析中医治疗骨质疏松症的机制。方法将大鼠分为正常组、诱导组、补肾组、健脾组、补肾健脾组(随机数字表法),制备含药血清,选取第4代BMSCs进行实验,运用免疫荧光法分析各组成肌分化情况,酶联免疫吸附法检测TNF-α、JNK蛋白表达量。结果①BMSCs经诱导15d后,通过免疫荧光化学染色法检测MyoG荧光平均强度,结果显示:各组均显著高于正常组(P<0.01),补肾健脾组最高。②TNF-α蛋白检测:补肾组、补肾健脾组显著低于正常组(P<0.01)。③JNK蛋白检测:各组显著低于正常组(P<0.01)。结论①各治法中,补肾健脾法对大鼠BMSCs成肌分化的促进效果最佳,为通过脾肾、肌骨双方向治疗OP提供可能。②各中医治法均对大鼠BMSCs的成肌分化存在促进作用,这可能与降低TNF-α、JNK水平有关。

Effect of Sijunzi Decoction on the Myonuclear Domain of Rat Soleus in Spleen Qi Deficiency

Objective: To study the mechanism of Sijunzi decoction treating limb weakness in spleen Qi deficiency (SQD) based on the myonuclear domain (MND) theory. Methods: 40 male Sprague-Dawley rats were randomly divided into the normal group, SQD model group (model group), SQD+ still water group (SW group) and SQD+ Sijunzi decoction group (CM group), 10 rats each group;Grip-Strength Meter was used to measure limb grip strength;transmission electron microscope was employed to observe the ultrastructural changes of the myofibers, Image Pro 6.0 was used to measure the myonuclear numbers, cross-section area (CSA) and then their ratios (the MND sizes) were calculated, immunofluorescence assay was chosen to test the expressions of paired box gene 7 (Pax7) and myogenic differentiation antigen (MyoD). Results: Compared with those in the normal group, limb grip strength was decreased, sarcomeres were abnormal, and all the myonuclear numbers, CSA and MND sizes were reduced, but the Pax7+ cell numbers were increased, significantly, in the model and SW groups;Compared with those in the model and SW groups, limb grip strength was increased, sarcomeres were basically normal, the myonuclear number and CSA were both greater, and the Pax7+ and MyoD+ cell numbers were both increased, significantly, in the CM group. Conclusion: Sijunzi decoction might increase the myonuclear number by activating the MSCs to treat limb weakness in SQD.

艾灸联合免疫检查点抑制剂与化疗治疗晚期非鳞非小细胞肺癌临床研究

目的:观察艾灸联合免疫检查点抑制剂与化疗治疗肺脾气虚型晚期非鳞非小细胞肺癌的临床疗效。方法:选取62例肺脾气虚型晚期非鳞非小细胞肺癌患者,按照随机数字表法分成对照组与试验组各31例。对照组予免疫检查点抑制剂与化疗治疗,直至疾病进展或发生不可耐受的毒性反应,化疗期间予常规抑酸护胃、止吐、护肝等对症支持治疗。试验组在对照组基础上给予艾灸治疗。比较2组中医证候积分,中医证候疗效,实体瘤疗效,无进展生存时间(PFS),CD3^(+)、CD4^(+)、CD8^(+)T淋巴细胞百分比,CD4^(+)/CD8^(+)值,以及药物不良反应。结果:治疗后,对照组中医证候积分与同组治疗前比较,差异无统计学意义(P>0.05);试验组中医证候分较治疗前降低,且低于对照组(P<0.05)。试验组与对照组中医证候改善率分别为77.42%、41.94%,2组比较,差异有统计学意义(P<0.05)。试验组与对照组肿瘤总缓解率(ORR)分别为58.06%、51.61%,疾病控制率(DCR)分别为83.87%、77.41%,2组ORR、DCR比较,差异均无统计学意义(P>0.05)。试验组PFS长于对照组(Log Rank P<0.05)。治疗方式为影响晚期非鳞非小细胞肺癌PFS的独立因素,与对照组比较,试验组疾病进展风险较低[HR=0.57 (95%CI 0.33~0.99)]。治疗后,试验组CD3^(+)、CD4^(+)T淋巴细胞百分比及CD4^(+)/CD8^(+)值均较治疗前升高,CD8^(+)T淋巴细胞百分比均较治疗前下降,差异均有统计学意义(P<0.05);对照组CD3^(+)、CD8^(+)T淋巴细胞百分比均较治疗前下降,差异均有统计学意义(P<0.05);对照组CD4^(+)T淋巴细胞百分比及CD4^(+)/CD8^(+)值治疗前后比较,差异均无统计学意义(P>0.05);试验组CD3^(+)、CD4^(+)T淋巴细胞百分比及CD4^(+)/CD8^(+)值均高于对照组(P<0.05)。2组白细胞减少、中性粒细胞减少、贫血、血小板减少、肝功能异常、肾功能异常、恶心呕吐、反应性毛细血管增生、乏力、食欲下降、免疫性肺炎总发生率及上述不良反应程度≥3级发生率比较,差异均无统计学意义(P>0.05)。结论:艾灸联合免疫检查点抑制剂与化疗治疗能改善肺脾气虚型晚期非鳞非小细胞肺癌的临床症状,提高其免疫功能和延长PFS。

“脾肾相关”治法SDF-1/CXCR4通路对BMSCs成肌、MDSCs成骨分化的影响

目的探究SDF-1/CXCR4通路在补肾健脾法干预下对骨髓间充质干细胞(BMSCs)成肌、肌源性干细胞(MDSCs)成骨分化的影响。方法正常、诱导(正常血清加相应成骨、成肌诱导液)、补肾阴、补肾阳、健脾、补肾健脾血清干预BMSCs成肌、MDSCs成骨分化培养15 d后取材。免疫荧光鉴定Desmin表达,Image J分析细胞平均荧光强度;茜素红染色观察成骨分化矿化结节;酶联免疫吸附法(ELISA)检测Myosin、BMP2、以及SDF-1、CXCR4蛋白表达;实时荧光定量聚合酶链式反应(Real-time qPCR)检测细胞SDF-1、CXCR4 mRNA表达。结果与正常组相比,BMSCs成肌分化中,Myosin蛋白水平降低(P<0.05)、诱导组Desmin表达升高(P<0.01)、SDF-1、CXCR4蛋白及mRNA表达升高;MDSCs成骨分化中,诱导组出现较小面积的矿化结节,BMP2蛋白表达升高(P<0.05)、SDF-1、CXCR4蛋白及mRNA表达升高。与诱导组相比,BMSCs成肌分化中,用药组Desmin表达升高(P<0.01),Myosin蛋白水平升高(P<0.05)、SDF-1和CXCR4蛋白及mRNA表达水平显著升高;MDSCs成骨分化中,用药组矿化结节数量增多、面积增大,BMP2蛋白水平升高(P<0.05)、SDF-1和CXCR4蛋白及mRNA表达升高。结论补肾健脾中医不同治法可提高SDF-1/CXCR4通路蛋白及mRAN表达从而促进BMSCs成肌、MDSCs成骨分化,补肾健脾法促进作用最为明显。

基于脾-线粒体相关性探讨ICC功能障碍

功能性胃肠病(Functional gastrointestinal disorders,FGIDs)症状重叠常常存在病情错综复杂、临床中诊疗困难、易加重患者的心理、经济负担及造成相应的医疗资源的消耗等现状。研究发现,功能性消化不良(Functional dyspepsia,FD)和胃食管反流病(Gastroesophageal reflux disease,GERD)可能存在潜在的病理生理学联系,目前主要认为与食管下括约肌松弛及胃排空延迟所致胃肠道动力障碍有关。脾为后天之本,气血生化之源,布散精微于周身,蕴含机体所需能量及物质。线粒体有“动力工厂”之称,通过三羧酸循环及氧化磷酸化生成ATP为机体生命活动提供能量的同时还可以生成血红素。脾与线粒体的功能体现了相同生命活动的两种不同诠释方式。相关研究认为,辨证为脾虚时线粒体能量代谢障碍所致的Cajal间质细胞(Interstitial cells of Cajal,ICC)功能障碍与胃肠动力障碍关系密切。故本文基于“脾与线粒体”相关性,探讨ICC功能障碍与FD-GERD症状重叠的关系,提出脾虚与线粒体能量代谢障碍引发ICC功能障碍是造成FDGERD症状重叠的重要因素。除此之外,从通降理论入手,可以为诊治FD-GERD症状重叠提供较高的临床实用价值。

肝纤维化进展中脾脏对肝脏巨噬细胞活化肝星状细胞作用的影响

目的 研究肝纤维化小鼠脾脏对肝脏中巨噬细胞活化肝星状细胞作用的影响。方法 18只雄性C57BL/6小鼠随机分为3组,A、B组注射CCl4制备肝纤维化模型,C组注射玉米油作为正常对照组。4周后,A、B组分别行脾切除术(切脾组)或假手术(有脾组)。继续注射2周后取材3组小鼠肝脏,制备肝脏匀浆(L-Homo)并分离肝脏细胞。Luminex检测3组L-Homo中IL-1β、IL-13、TGF-β、TNF-α、PDGF-β、VEGF的表达;RT-qPCR与流式细胞术进一步观察如上因子在A、B组肝脏巨噬细胞(L-Mφ)及肝脏其他单个核细胞中的表达。利用A/B组L-Homo分别进行体外处理,以模拟脾脏存在与否时肝脏微环境对巨噬细胞的影响。收集接受不同L-Homo处理的巨噬细胞,一方面利用RT-qPCR比较其中细胞因子与谷氨酰胺合成/分解酶与谷氨酰胺转运蛋白的表达差异;另一方面与肝星状细胞JS1进行共培养,分析其对JS1存活与胞外基质表达的影响。组间比较使用Student’s t检验(两组间)或单因素方差分析(多组间)。结果 与正常对照组相比,IL-1β、IL-13、TGF-β与TNF-α的浓度在模型组L-Homo中显著升高,且在模型有脾组中显著高于切脾组;其中巨噬细胞是表达这些细胞因子的主要细胞类型群。相较于切脾组,有脾组L-Homo体外处理上调巨噬细胞中IL-1β、TGF-β、TNF-α等细胞因子以及谷氨酰胺酶的表达,并且促进巨噬细胞发挥活化肝星状细胞胞外基质分子表达的能力。结论 脾脏参与调控L-Mφ炎性因子表达,增强其活化肝星状细胞的能力。

基于“肝病实脾”理论探讨逍遥散对肝纤维化大鼠肝星状细胞铁死亡的影响

目的:基于“肝病实脾”理论探讨逍遥散对肝纤维化大鼠肝星状细胞铁死亡的影响。方法:采用腹腔注射四氯化碳的方法建立肝纤维化大鼠模型,随机分为模型组、逍遥散组、水飞蓟宾葡甲胺组,每组12只,另选12只SD大鼠腹腔注射等剂量玉米油设为对照组,以逍遥散、水飞蓟宾葡甲胺分组处理后检测各组大鼠肝功能指标天冬氨酸氨基转移酶(AST)和丙氨酸氨基转移酶(ALT);检测各组大鼠肝指数并以Masson染色检测各组大鼠肝纤维化,比较其肝胶原容积分数(CVF);以免疫组织化学染色检测各组大鼠肝星状细胞活化,比较其肝组织α-SMA阳性表达;通过16SrRNA基因测序检测各组大鼠肠道菌群改变。分离培养上述各组大鼠原代肝星状细胞(HSC),以试剂盒测定各组大鼠HSC铁死亡指标:铁含量、谷胱甘肽(GSH)及丙二醛(MDA)水平;以免疫印迹法检测各组大鼠HSC铁死亡标志蛋白[长链脂酰辅酶A合成酶4(ACSL4)、环加氧酶2(PTGS2)、谷胱甘肽过氧化酶4(GPX4)]表达。结果:与对照组相比,模型组大鼠拟杆菌目丰度、HSC铁含量、MDA水平、ACSL4与PTGS2蛋白表达均明显降低(P<0.05),血清AST与ALT水平、肝指数、肝CVF、肝组织α-SMA阳性比、ACE指数、梭菌目丰度、HSC中GSH水平与GPX4蛋白表达均明显升高(P<0.05);与模型组相比,逍遥散组、水飞蓟宾葡甲胺组大鼠拟杆菌目丰度、HSC铁含量、MDA水平、ACSL4与PTGS2蛋白表达均升高(P<0.05),血清AST与ALT水平、肝指数、肝CVF、肝组织α-SMA阳性比、ACE指数、梭菌目丰度、HSC中GSH水平与GPX4蛋白表达均降低(P<0.05);与逍遥散组相比,水飞蓟宾葡甲胺组大鼠各指标差异无统计学意义(P>0.05)。结论:逍遥散可通过诱导肝星状细胞铁死亡而抑制其活性,进而减轻肝纤维化大鼠肝组织肝纤维化,减轻其肝功能损伤,最终对大鼠发挥明显肝保护作用。