<p>PDF file - 204KB, Supplementary Table 1. Clinicopathologic characteristics of lung adenocarcinoma patients. Supplementary Table 2. Correlation between the clinicopathological features and expression of miR-483-5p. Supplementary Table 3. Correlation between the tumor grades and the expression of proteins. Supplementary Table 4. Oligonucleotides used in this study.</p>

PDF file - 488KB, Supplementary Figure 1. Upregulation of miR-483-5p is correlated with the progression of human lung adenocarcinoma. Supplementary Figure 2. miR-483-5p did not affect lung tumor growth in vitro and in vivo. Supplementary Figure 3. miR-483-5p, but not miR-483-3p promotes lung cancer cell EMT in vitro. Supplementary Figure 4. Identification of putative miR-483-5p target genes in A549 cells by 2D-DGE. Supplementary Figure 5. Knockdown of ALCAM did not affect the expression levels of the EMT-related proteins.

该文旨在分析基于云班课混合式教学在医学细胞生物学课程中的教学效果.选取南方医科大学十个专业的2019级和2020级本科生为研究对象,实验组(2020级)采用以传统授课为主,云班课为辅的混合式教学;对照组(2019级)采用传统课堂教学;课程结束后,以期末考试成绩和收集的实验组教学效果调查问卷为统计分析的基本资料.实验组期末成绩的平均值为74.21分,对照组为72.40分;实验组期末成绩及格率为91％,对照组及格率为85％,具有统计学差异(P＜0.05).问卷调查结果显示,实验组学生对云班课混合式教学方法的认同程度高.将基于云班课混合式教学的方法应用于医学细胞生物学教学能够在一定程度上提升教学效果及学生的学习兴趣.

BACKGROUND:Specific immunotherapy, including agonists for Toll-like receptor 2 (TLR2), have been shown to protect from allergies and to have a high immunomodulatory capacity.METHODS:A new antibody, TSP-2, reactive against an epitope of the extracellular domain of TLR2, was identified. The effect of the antibody on dendritic cells was assessed by immunohistochemistry, Western blot, and flow cytometric analysis. The effect of TSP-2 in a murine asthma model induced with ovalbumin (OVA) was assessed. The model is a form of airway hyperresponsiveness (AHR) and was analyzed by whole-body plethysmography, the measurement of Th1/Th2 cytokines in bronchial alveolar lavage fluid (BALF) and serum by ELISA, and the CCK-8 assay for lymphocyte proliferation. The effect of TSP-2 on the maturation of bone marrow-derived dendritic cells (BMDCs) was assessed by flow cytometric analysis.RESULTS:TSP-2 promoted the maturation of dendritic cells and the proliferation of lymphocyte in vitro and in vivo. The effect of TSP-2 on T helper 1 (Th1)/Th2 cytokine secretion was slightly more powerful than that of Pam3CSK4. TSP-2 antibody reduced AHR and OVA-specific IgE levels in allergic asthma. TSP-2 antibody also reduced lung inflammation and decreased leukocyte numbers in an OVA-sensitized and challenged asthma model. TSP-2 antibody increased OVA-stimulated I-A, CD80, CD86, and MHC-II levels on BMDCs.CONCLUSIONS:This study identifies a novel therapeutic strategy for AHR, which uses antibodies reactive against TLR2. It also provides theoretical evidence for the control of allergic asthma by targeting TLR2.

The small GTPase ras homolog enriched in brain ( Rheb ) is a downstream target of tuberous sclerosis complex 1/2 ( TSC1/2 ) and an upstream activator of the mechanistic target of rapamycin complex 1 (mTORC1), the emerging essential modulator of M1/M2 balance in macrophages. However, the role and regulatory mechanisms of Rheb in macrophage polarization and allergic asthma are not known. In the present study, we utilized a mouse model with myeloid cell-specific deletion of the Rheb1 gene and an ovalbumin (OVA)-induced allergic asthma model to investigate the role of Rheb1 in allergic asthma and macrophage polarization. Increased activity of Rheb1 and mTORC1 was observed in myeloid cells of C57BL/6 mice with OVA-induced asthma. In an OVA-induced asthma model, Rheb1 -KO mice demonstrated a more serious inflammatory response, more mucus production, enhanced airway hyper-responsiveness, and greater eosinophil numbers in bronchoalveolar lavage fluid (BALF). They also showed increased numbers of bone marrow macrophages and BALF myeloid cells, elevated M2 polarization and reduced M1 polarization of macrophages. Thus, we have established that Rheb1 is critical for the polarization of macrophages and inhibition of allergic asthma. Deletion of Rheb1 enhances M2 polarization but decreases M1 polarization in alveolar macrophages, leading to the aggravation of OVA-induced allergic asthma.

Loss of Tsc1/Tsc2 results in excess cell growth that eventually forms hamartoma in multiple organs. Our study using a mouse model with Tsc1 conditionally knockout in mammary epithelium showed that Tsc1 deficiency impaired mammary development. Phosphorylated S6 was up-regulated in Tsc1 −/− mammary epithelium, which could be reversed by rapamycin, suggesting that mTORC1 was hyperactivated in Tsc1 −/− mammary epithelium. The mTORC1 inhibitor rapamycin restored the development of Tsc1 −/− mammary glands whereas suppressed the development of Tsc1 wt/wt mammary glands, indicating that a modest activation of mTORC1 is critical for mammary development. Phosphorylated PDK1 and AKT, nuclear ERα, nuclear IRS-1, SGK3 and cell cycle regulators such as Cyclin D1, Cyclin E, CDK2, CDK4 and their target pRB were all apparently down-regulated in Tsc1 −/− mammary glands, which could be reversed by rapamycin, suggesting that suppression of AKT by hyperactivation of mTORC1, inhibition on nuclear ERα signaling and down-regulation of cell-cycle-driving proteins play important roles in the retarded mammary development induced by Tsc1 deletion. This study demonstrated for the first time the in vivo role of Tsc1 in pubertal mammary development of mice and revealed that loss of Tsc1 does not necessarily lead to tissue hyperplasia.

Astrocytes respond to CNS insults through reactive astrogliosis, but the underlying mechanisms are unclear. In this study, we show that inactivation of mechanistic target of rapamycin complex (mTORC1) signaling in postnatal neurons induces reactive astrogliosis in mice. Ablation of Raptor (an mTORC1-specific component) in postmitotic neurons abolished mTORC1 activity and produced neurons with smaller soma and fewer dendrites, resulting in microcephaly and aberrant behavior in adult mice. Interestingly, extensive astrogliosis without significant astrocyte proliferation and glial scar formation was observed in these mice. The inhibition of neuronal mTORC1 may activate astrogliosis by reducing neuron-derived fibroblast growth factor 2 (FGF-2), which might trigger FGF receptor signaling in astrocytes to maintain their nonreactive state, and FGF-2 injection successfully prevented astrogliosis in Raptor knock-out mice. This study demonstrates that neuronal mTORC1 inhibits reactive astrogliosis and plays an important role in CNS pathologies.

Previous studies have reported that mTORC2 promotes cell survival through phosphorylating AKT and enhancing its activity. We reveal another mechanism by which mTORC2 controls apoptosis. Inactivation of mTORC2 promotes binding of CIP2A to PP2A, leading to reduced PP2A activity toward c-Myc serine 62 and, consequently, enhancement of c-Myc phosphorylation and expression. Increased c-Myc activity induces transcription of pri-miR-9-2/miR-9-3p, in turn inhibiting expression of E2F1, a transcriptional factor critical for cancer cell survival and tumor progression, resulting in enhanced apoptosis. In vivo experiments using B cell-specific mTORC2 (rapamycin-insensitive companion of mTOR) deletion mice and a xenograft tumor model confirmed that inactivation of mTORC2 causes up-regulation of c-Myc and miR-9-3p, down-regulation of E2F1, and consequent reduction in cell survival. Conversely, Antagomir-9-3p reversed mTORC1/2 inhibitor-potentiated E2F1 suppression and resultant apoptosis in xenograft tumors. Our in vitro and in vivo findings collectively demonstrate that mTORC2 promotes cell survival by stimulating E2F1 expression through a c-Myc- and miR-9-3p-dependent mechanism.

南方医科大学自2005年开始共招收10届留学生。细胞生物学教研室承担了留学生细胞生物学全英教学任务，并取得了较好的教学效果。文章从不断提高师资力量、精心选择教材、运用多种教学手段、重视实验课教学四个主要方面总结了经验教训，并期望进一步探索留学生细胞生物学教学改革方法，不断提高留学生教学质量。

目的 改进洋葱内表皮细胞中DNA和RNA的染色方法. 方法 使用固定液固定后,以不同浓度的甲基绿派洛宁混合染色液对洋葱内表皮细胞进行染色处理,与传统方法对比,观察染色效果. 结果 用甲醇冰醋酸固定液固定后,洋葱内表皮细胞不会发生质壁分离,更容易染色;甲基绿与派洛宁的浓度比为3∶2和1∶1时能在细胞中更加分明地将细胞核染成蓝绿色,将细胞质和核仁染成红色. 结论 用甲醇冰醋酸固定液固定,甲基绿与派洛宁的浓度比为3∶2至1∶1时能得到较好的实验效果.

The nodal regulatory properties of microRNAs (miRNA) in metastatic cancer may offer new targets for therapeutic control. Here, we report that upregulation of miR-483-5p is correlated with the progression of human lung adenocarcinoma. miR-483-5p promotes the epithelial–mesenchymal transition (EMT) accompanied by invasive and metastatic properties of lung adenocarcinoma. Mechanistically, miR-483-5p is activated by the WNT/b-catenin signaling pathway and exerts its prometastatic function by directly targeting the Rho GDP dissociation inhibitor alpha (RhoGDI1) and activated leukocyte cell adhesion molecule (ALCAM), two putative metastasis suppressors. Furthermore, we found that downregulation of RhoGDI1 enhances expression of Snail, thereby promoting EMT. Importantly, miR-483-5p levels are positively correlated with b-catenin expression, but are negatively correlated with the levels of RhoGDI1 and ALCAM in human lung adenocarcinoma. Our findings reveal that miR-483-5p is a critical b-catenin–activated prometastatic miRNA and a negative regulator of the metastasis suppressors RhoGDI1 and ALCAM. Cancer Res; 74(11); 3031–42. 2014 AACR.

Abstract The nodal regulatory properties of microRNAs (miRNA) in metastatic cancer may offer new targets for therapeutic control. Here, we report that upregulation of miR-483-5p is correlated with the progression of human lung adenocarcinoma. miR-483-5p promotes the epithelial–mesenchymal transition (EMT) accompanied by invasive and metastatic properties of lung adenocarcinoma. Mechanistically, miR-483-5p is activated by the WNT/β-catenin signaling pathway and exerts its prometastatic function by directly targeting the Rho GDP dissociation inhibitor alpha (RhoGDI1) and activated leukocyte cell adhesion molecule (ALCAM), two putative metastasis suppressors. Furthermore, we found that downregulation of RhoGDI1 enhances expression of Snail, thereby promoting EMT. Importantly, miR-483-5p levels are positively correlated with β-catenin expression, but are negatively correlated with the levels of RhoGDI1 and ALCAM in human lung adenocarcinoma. Our findings reveal that miR-483-5p is a critical β-catenin–activated prometastatic miRNA and a negative regulator of the metastasis suppressors RhoGDI1 and ALCAM. Cancer Res; 74(11); 3031–42. ©2014 AACR.

Mutation of TSC (encoding tuberous sclerosis complex protein) and activation of mammalian target of rapamycin (mTOR) have been implicated in the pathogenesis of several renal diseases, such as diabetic nephropathy and polycystic kidney disease. However, the role of mTOR in renal potassium excretion and hyperkalemia is not known. We showed that mice with collecting-duct (CD)-specific ablation of TSC1 (CDTsc1KO) had greater mTOR complex 1 (mTORC1) activation in the CD and demonstrated features of pseudohypoaldosteronism, including hyperkalemia, hyperaldosteronism, and metabolic acidosis. mTORC1 activation caused endoplasmic reticulum stress, columnar cell lesions, and dedifferentiation of CD cells with loss of aquaporin-2 and epithelial-mesenchymal transition-like phenotypes. Of note, mTORC1 activation also reduced the expression of serum- and glucocorticoid-inducible kinase 1, a crucial regulator of potassium homeostasis in the kidney, and decreased the expression and/or activity of epithelial sodium channel-α, renal outer medullary potassium channel, and Na(+), K(+)-ATPase in the CD, which probably contributed to the aldosterone resistance and hyperkalemia in these mice. Rapamycin restored these phenotypic changes. Overall, this study identifies a novel function of mTORC1 in regulating potassium homeostasis and demonstrates that loss of TSC1 and activation of mTORC1 results in dedifferentiation and dysfunction of the CD and causes hyperkalemia. The CDTsc1KO mice provide a novel model for hyperkalemia induced exclusively by dysfunction of the CD.

探索在基础医学专业研究生培养中,如何加强研究生教学能力的培养,其具体实施过程包括安排研究生参加本科生实验教学、本科生实习带教、组织中期考核的理论课试讲及定期学术汇报。加强教学能力培养是提高研究生整体素质,增加就业竞争力的有效途径。

The stem cell-based experimental therapies are partially successful for the recovery of spinal cord injury (SCI). Recently, acellular spinal cord (ASC) scaffolds which mimic native extracellular matrix (ECM) have been successfully prepared. This study aimed at investigating whether the spinal cord lesion gap could be bridged by implantation of bionic-designed ASC scaffold alone and seeded with human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) respectively, and their effects on functional improvement. A laterally hemisected SCI lesion was performed in adult Sprague–Dawley (SD) rats (n=36) and ASC scaffolds seeded with or without hUCB-MSCs were implanted into the lesion immediately. All rats were behaviorally tested using the Basso–Beattie–Bresnahan (BBB) test once a week for 8weeks. Behavioral analysis showed that there was significant locomotor recovery improvement in combined treatment group (ASC scaffold and ASC scaffold+hUCB-MSCs) as compared with the SCI only group (p<0.01). 5-Bromodeoxyuridine (Brdu)-labeled hUCB-MSCs could also be observed in the implanted ACS scaffold two weeks after implantation. Moreover, host neural cells (mainly oligodendrocytes) were able to migrate into the graft. Biotin–dextran–amine (BDA) tracing test demonstrated that myelinated axons successfully grew into the graft and subsequently promoted axonal regeneration at lesion sites. This study provides evidence for the first time that ASC scaffold seeded with hUCB-MSCs is able to bridge a spinal cord cavity and promote long-distance axon regeneration and functional recovery in SCI rats.

Thymosin alpha-1 (Tα1) has been used as an immune potentiator for treatment of immune deficiency diseases by injection administration. However, injection is inconvenient and may cause many side effects. In order to improve the administration convenience of Tα1, a human Tα1 gene transformed Bifidobacterium longum (BL-Tα1) was prepared and its effects on mice immunity by oral administration were investigated. The Balb/c mice were treated with BL-Tα1, which was pre-induced with 0.2% l-arabinose, every other day for 2weeks. The B. longum transformed with empty vector (BL-0) was used as the negative control, and normal saline (NS, 0.9% saline) was used as the blank control. The results shown that (1) the CD3+CD4+ and CD3+CD8+ T-cells in blood, spleen and thymus, and the CD4+CD8+ cells in thymus and spleen of BL-Tα1 group were all significantly increased than that of negative control BL-0 group respectively; (2) the interferon-γ (IFN-γ) and interleukin-12 (IL-12) in serum of BL-Tα1 group were significantly increased. No significant differences were found in the levels of tumor necrosis factor-α (TNF-α) and interleukin-4 (IL-4) between BL-Tα1 group and BL-0 group; (3) thymic hyperplasia and lymphadenectasis were observed in BL-Tα1 group after three-month treatment. In conclusion, the Tα1-transformed B. longum promotes thymus and lymph nodes growth, stimulates T cell proliferation and maturation, and enhances cellular immunity through Th1 pathway by oral administration.

Objective To observe the clinical efficacy of"Xiehuo Zhitong Decoction"in treating migraine of hyperactivity of liver fire pattern.Methods Sixtyfour cases were randomized into two groups: treatment group in which 33 cases were treated with"Xiehuo Zhitong Decoction",and control group in which 31 cases were treated with Flunarizine Hydrochloride,with the course of 8 weeks.The clinical efficacy and migraine score were observed.Results The overall effective rate was 87.88% in the treatment group and 74.19% in the control group,with a difference between the two groups(P < 0.05);after treatment,the migraine score in the two groups were reduced significantly(P < 0.01),but without significant difference between the two groups(P > 0.05).Conclusion "Xiehuo Zhitong Decoction"is quite effective in treating migraine in type of hyperactivity of liver fire.

mTORC2, the mammalian target of rapamycin complex 2 is activated by upstream growth factors, and performs two major functions, phosphorylation of AKT at the serine of 473 and cell cycle-dependent organization of actin cytoskeleton. However, the mechanisms through which mTORC2 is triggered by these signals remain unclear. We demonstrated, for the first time, that inhibitor of nuclear factor κ-B kinase (IKK) interacted with rictor and regulated mTORC2 activity. Not only endogenously, but ectopically expressed IKK α and IKK β physically interacted with rictor. An in vitro binding assay revealed that rictor interacted with IKKα and IKKβ from amino acids 999 to 1397. Moreover, chemical inhibition of IKK, knockdown of IKK by small interference RNA (siRNA), or ectopic expression of kinase-dead IKK (IKK KD) repressed phosphorylation of AKT (S473) in a variety of cell lines and decreased the kinase activity of mTORC2. In NIH 3T3 cells, inhibition of IKK also reduced phosphorylation of protein kinase α (PKCα) (S657) and resulted in disorganization of actin cytoskeleton. Interestingly, the interaction between IKKα/β and rictor was increased, while the mTOR-rictor association was attenuated by inhibition of IKK. We identified a novel signaling mechanism for the regulation of mTORC2 by IKK: IKK interacted with rictor and regulated the function of mTORC2 including phosphorylation of AKT (S473) and organization of actin cytoskeleton. Inactivated IKK interacted with rictor and competed against mTOR, which resulted in a reduced mTORC2 level and a decrease in mTORC2 activity.

Most of breast cancers are resistant to mammalian target of rapamycin complex 1 (mTORC1) inhibitors rapamycin and rapalogs. Recent studies indicate mTORC2 is emerging as a promising cancer therapeutic target. In this study, we compared the inhibitory effects of targeting mTORC1 with mTORC2 on a variety of breast cancer cell lines and xenograft. We demonstrated that inhibition of mTORC1/2 by mTOR kinase inhibitors PP242 and OSI-027 effectively suppress phosphorylation of Akt (S473) and breast cancer cell proliferation. Targeting of mTORC2 either by kinase inhibitors or rictor knockdown, but not inhibition of mTORC1 either by rapamycin or raptor knockdown promotes serum starvation- or cisplatin-induced apoptosis. Furthermore, targeting of mTORC2 but not mTORC1 efficiently prevent breast cancer cell migration. Most importantly, in vivo administration of PP242 but not rapamycin as single agent effectively prevents breast tumor growth and induces apoptosis in xenograft. Our data suggest that agents that inhibit mTORC2 may have advantages over selective mTORC1 inhibitors in the treatment of breast cancers. Given that mTOR kinase inhibitors are in clinical trials, this study provides a strong rationale for testing the use of mTOR kinase inhibitors or combination of mTOR kinase inhibitors and cisplatin in the clinic.