Involvement of Cytokines and Hormones in the Development of Spermatogenesis In Vitro from Spermatogonial Cells of Cyclophosphamide-Treated Immature Mice

Aggressive chemotherapy treatment may lead to male infertility. Prepubertal boys do not produce sperm at this age, however, they have spermatogonial stem cells in their testes. Here, we examined the effect of intraperitoneal injection of cyclophosphamide (CP) on the capacity of immature mice (IM) to develop spermatogenesis in vivo and in vitro [using methylcellulose culture system (MCS)]. Our results show a significant decrease in testicular weight, total number of testicular cells, and the number of Sertoli, peritubular, premeiotic, and meiotic/post-meiotic cells, but an increase in the percentages of damaged seminiferous tubules in CP-treated IM compared to control. The functionality of Sertoli cells was significantly affected. The addition of testosterone to isolated cells from seminiferous tubules of CP-treated IM significantly increased the percentages of premeiotic (CD9-positive cells) and meiotic/post-meiotic cells (ACROSIN-positive cells) developed in MCS compared to control. The addition of FSH did not affect developed cells in MCS compared to control, but in combination with testosterone, it significantly decreased the percentages of CD9-positive cells and ACROSIN-positive cells. The addition of IL-1 did not affect developed cells in MCS compared to control, but in combination with testosterone, it significantly increased the percentages of VASA-positive cells and BOULE-positive cells compared to IL-1 or testosterone. Addition of TNF significantly increased only CD9-positive cells in MCS compared to control, but in combination with testosterone, it significantly decreased ACROSIN-positive cells compared to testosterone. Our results show a significant impairment of spermatogenesis in the testes of CP-treated IM, and that spermatogonial cells from these mice proliferate and differentiate to meiotic/post-meiotic cells under in vitro culture conditions.     

Molecular Iodine/Cyclophosphamide Synergism on Chemoresistant Neuroblastoma Models

Neuroblastoma (Nb), the most common extracranial tumor in children, exhibited remarkable phenotypic diversity and heterogeneous clinical behavior. Tumors with MYCN overexpression have a worse prognosis. MYCN promotes tumor progression by inducing cell proliferation, de-differentiation, and dysregulated mitochondrial metabolism. Cyclophosphamide (CFF) at minimum effective oral doses (metronomic therapy) exerts beneficial actions on chemoresistant cancers. Molecular iodine (I₂) in coadministration with all-trans retinoic acid synergizes apoptosis and cell differentiation in Nb cells. This work analyzes the impact of I₂ and CFF on the viability (culture) and tumor progression (xenografts) of Nb chemoresistant SK-N-BE(2) cells. Results showed that both molecules induce dose-response antiproliferative effects, and I₂ increases the sensibility of Nb cells to CFF, triggering PPARγ expression and acting as a mitocan in mitochondrial metabolism. In vivo oral I₂/metronomic CFF treatments showed significant inhibition in xenograft growth, decreasing proliferation (Survivin) and activating apoptosis signaling (P53, Bax/Bcl-2). In addition, I₂ decreased the expression of master markers of malignancy (MYCN, TrkB), vasculature remodeling, and increased differentiation signaling (PPARγ and TrkA). Furthermore, I₂ supplementation prevented loss of body weight and hemorrhagic cystitis secondary to CFF in nude mice. These results allow us to propose the I₂ supplement in metronomic CFF treatments to increase the effectiveness of chemotherapy and reduce side effects.     

非变性I型胶原蛋白对免疫低下大鼠免疫功能的调节作用

该研究旨在观察非变性I型胶原蛋白（Non-denatured type-I collagen,NDC-I）对免疫低下模型大鼠免疫功能的调节作用。动物实验采用腹腔注射环磷酰胺法建立免疫低下大鼠模型,观察NDC-I对大鼠的胸腺指数和脾脏指数,白细胞计数和分类,血清中免疫球蛋白A（IgA）、免疫球蛋白G（IgG）、白介素-2（IL-2）、白介素-4（IL-4）、白介素-10（IL-10）、干扰素-γ（IFN-γ）和肿瘤坏死因子α（TNF-α）,脾脏和胸腺的乳酸脱氢酶（LDH）活性和组织形态学变化。结果显示,在给予NDC-I干预30 d后,与模型组比较,NDC-I组胸腺指数上升（p<0.05）,白细胞计数显著提高（p<0.01）,血清中IgA、IgG、IL-2、IL-4、IL-10、IFN-γ和TNF-α含量为43.32 µg/mL、283.32 µg/mL、1827.17 ng/L、135.97 pg/mL、113.87 ng/L、2302.44 pg/mL、469.91 ng/L,水平显著升高（p<0.01）,脾脏中LDH活性显著上升（p<0.01）,脾脏和胸腺组织病理学损伤减轻。结果证明,NDC-I对环磷酰胺致免疫低下大鼠的免疫功能具有一定的调节作用,也为胶原蛋白等相关功能产品的综合开发与充分利用提供了参考依据。     

同胞相合异基因造血干细胞移植治疗恶性血液病的疗效

目的探讨同胞相合异基因造血干细胞移植治疗恶性血液病的临床疗效。方法将26例恶性血液病患者按移植前疾病状态分为2组：高危组11例和标危组15例。2组患者均采用外周血同胞相合异基因造血干细胞移植。预处理方案：高危组采用全身照射＋环磷酰胺,标危组采用白消安＋环磷酰胺;移植物抗宿主病的预防：2组均采用环孢素A＋短程甲氨蝶呤,或加霉酚酸脂。观察2组患者造血重建和急、慢性移植物抗宿主病、巨细胞病毒活动性感染/巨细胞病毒病、移植复发及移植相关死亡及无病生存等情况。结果 2组患者移植后均获得造血重建,均为完全供体嵌合型。2组患者移植后14例发生急性移植物抗宿主病（aGVHD）,累积发病率为53.8%（14/26）。9例发生慢性移植物抗宿主病（cGVHD）,累积发病率为45.0%（9/20）。2组患者移植后有20例（76.9%）出现巨细胞病毒血症,无一例患者发生巨细胞病毒病。标危组复发率与高危组比较差异无统计学意义（6.7%vs 18.2%,P〉0.05）。标危组移植相关病死率与高危组比较差异有统计学意义（13.3%vs 54.5%,P〈0.05）。高危组和标危组患者1、3年累积无病生存率分别为30.3%、85.1%和15.2%、68.1%（均P〈0.05）。结论移植前疾病状态是影响移植疗效的重要因素,移植物抗宿主病的发生对移植后疾病的复发及生活质量有重要的影响。     

小鼠肺组织中肿瘤坏死因子α水平的变化与真菌机会性感染的关系

目的探讨小鼠肺组织中肿瘤坏死因子α（tumor necrosis factor-α,TNF-α）水平的变化与肺组织真菌机会性感染的关系。方法将70只小鼠按数字表法分为2组：A组25只和B组45只。B组小鼠腹腔注射环磷酰胺（Cyclophosphamide,CY）80 mg.kg-1.d-1,A组小鼠腹腔注射等量的生理盐水。2组于8、24、48 h杀死小鼠,分离肺组织。Western Blot法检测2组小鼠肺组织胞浆中TNF-α的表达;比色法检测肺组织中髓过氧化物酶（My-eloperoxidase,MPO）的活性。A组小鼠注射无菌生理盐水24 h后、B组小鼠腹腔注射CY后8、24、48 h分别鼻吸入烟曲霉菌孢子,各组小鼠分别在感染24、96 h处死,匀浆肺组织培养,计数菌落数。结果 B组CY处理后8、24、48 h肺组织TNF-α水平分别为0.152±0.011、0.314±0.029和0.631±0.035,显著低于A组小鼠肺组织中TNF-α水平（0.812±0.043）（P〈0.01）。B组3个时间点的TNF-α水平比较差异有统计学意义（P〈0.01）。B组小鼠8、24、48 h肺组织中MPO活性均显著低于A组[（0.028±0.007）、（0.075±0.004）、（0.125±0.009）U.g-1vs（0.218±0.015）、（0.208±0.036）、（0.211±0.012）U.g-1,均P〈0.01]。A组烟曲霉菌菌落数在24、96 h时均显著低于B组（均P〈0.01）。结论小鼠肺组织TNF-α表达下调,降低了肺组织MPO活性水平,导致抗感染天然免疫抑制,是机体发生机会性真菌感染的机制之一。     

Processed Aloe vera Gel Ameliorates Cyclophosphamide-Induced Immunotoxicity

The effects of processed Aloe vera gel (PAG) on cyclophosphamide (CP)-induced immunotoxicity were examined in mice. Intraperitoneal injection of CP significantly reduced the total number of lymphocytes and erythrocytes in the blood. Oral administration of PAG quickly restored CP-induced lymphopenia and erythropenia in a dose-dependent manner. The reversal of CP-induced hematotoxicity by PAG was mediated by the functional preservation of Peyer’s patch cells. Peyer’s patch cells isolated from CP-treated mice, which were administered PAG, produced higher levels of T helper 1 cytokines and colony-stimulating factors (CSF) in response to concanavalin A stimulation as compared with those isolated from CP-treated control mice. PAG-derived polysaccharides directly activated Peyer’s patch cells isolated from normal mice to produce cytokines including interleukin (IL)-6, IL-12, interferon-γ, granulocyte-CSF, and granulocyte-macrophage-CSF. The cytokines produced by polysaccharide-stimulated Peyer’s patch cells had potent proliferation-inducing activity on mouse bone marrow cells. In addition, oral administration of PAG restored IgA secretion in the intestine after CP treatment. These results indicated that PAG could be an effective immunomodulator and that it could prevent CP-induced immunotoxic side effects.     

乌贼墨多糖缓解环磷酰胺致大鼠骨髓功能抑制作用的研究

探讨乌贼墨多糖(SIPS)对环磷酰胺所致骨髓功能抑制的缓解效应。以SD大鼠为模型,灌服SIPS和腹腔注射环磷酰胺共处理,检测大鼠外周血像和骨髓DNA含量。环磷酰胺不仅显著降低了大鼠外周血红细胞、白细胞和血小板的数量以及血红蛋白的含量,骨髓细胞DNA含量也被显著下调(p<0.01orp<0.05)。SIPS十分明显的弱化了该化疗药物的骨髓抑制功能,环磷酰胺对以上指标参数所导致的负效应均被该海洋活性物质不同程度的消减(p<0.05orp<0.01)。SIPS可缓解环磷酰胺所致大鼠骨髓造血功能抑制作用。     

酵母β-葡聚糖加锌复合配方对免疫抑制幼龄小鼠的免疫调节作用

目的:探究酵母β-葡聚糖加锌复合配方对环磷酰胺诱导免疫抑制幼龄小鼠的免疫调节作用。方法:48只3周龄BALB/c小鼠,随机分成空白组、模型组和复合物组。复合物组灌以由酵母β-葡聚糖和锌组成的复合物(相当于酵母β-葡聚糖:90 mg/kg/d、锌:2.8 mg/kg/d),空白组、模型组灌以等体积蒸馏水,模型组、复合物组在试验的第14、15 d以腹腔注射40 mg/kg环磷酰胺构建免疫低下模型,28 d后,再将小鼠分为两组,第Ⅰ组用于测定免疫器官指数、免疫细胞数目和活性以及细胞因子。第Ⅱ组用于血清溶血素实验和溶血空斑实验。结果:与模型组相比,酵母β-葡聚糖和柠檬酸锌复合配方能显著（P<0.05）提高小鼠免疫器官系数,促使脾淋巴细胞增殖和增加杯状细胞数量,极显著（P<0.01）提高半数溶血值（serum half hemolysis value, HC₅₀）、溶血空斑数、NK细胞活性,增加外周血中α肿瘤坏死因子（Tumor necrosis factor-α, TNF-α）、γ干扰素（interferon-γ, IFN-γ）浓度、白细胞数量,增加派氏结。结论:酵母β-葡聚糖加锌复合配方具有提高体液免疫、NK细胞活性,并增强肠黏膜免疫的作用。