目的探讨肺腺癌细胞恶性T细胞扩增序列1(MCTS1)介导骨髓源性抑制细胞(MDSCs)趋化的作用机制。方法利用生物信息学(http://timer.cistrome.org/#tab-8258-2)分析癌症基因组图谱(TCGA)中肺腺癌组织(n=515)MCTS1的表达水平与MDSCs浸润之间...目的探讨肺腺癌细胞恶性T细胞扩增序列1(MCTS1)介导骨髓源性抑制细胞(MDSCs)趋化的作用机制。方法利用生物信息学(http://timer.cistrome.org/#tab-8258-2)分析癌症基因组图谱(TCGA)中肺腺癌组织(n=515)MCTS1的表达水平与MDSCs浸润之间的相关性;利用IL-6+粒细胞-巨噬细胞集落刺激因子(GM-CSF)共同诱导人外周血单个核细胞(PBMCs)中MDSCs的生成,流式细胞术检测MDSCs生成比例,CD33磁珠分选MDSCs;收集肺腺癌SPC-A1细胞野生型以及MCTS1敲减和过表达SPC-A1细胞的无血清细胞上清液,利用Transwell小室进行MDSCs的趋化试验并计算趋化指数;羧基荧光素二醋酸盐琥珀酰亚胺酯(CFSE)标记CD8^(+)T细胞与发生趋化的MDSCs细胞共培养,流式细胞术检测CD8^(+)T细胞的增殖水平;实时荧光定量PCR(qRT-PCR)测定MDSCs相关趋化因子的mRNA水平;流式多因子芯片技术检测细胞上清液中C-X-C基序趋化因子配体1(CXCL1)的含量;Western blot检测janus激酶2(JAK2)的表达水平以及信号转导子和转录激活子1(STAT1)的活化水平。结果肺腺癌组织MCTS1与MDSCs浸润程度呈正相关(Spearmanρ=0.226,P<0.001);与空白对照组比较,IL-6和GM-CSF共同诱导PBMCs后可显著增加MDSCs的比例(4.95±1.03 vs 0.97±0.24,t=6.54,P<0.01);MCTS1过表达的SPC-A1细胞上清液对MDSCs的趋化指数较对照组显著升高(4.88±0.99 vs 2.94±0.27,q=6.29,P<0.01),而经MCTS1敲减的细胞上清液可使MDSCs的趋化指数显著降低(1.50±0.17 vs 2.94±0.27,q=4.69,P<0.05);MCTS1过表达的SPC-A1细胞中CXCL1 mRNA(2.79±0.16 vs 1.00±0.08,q=28.94,P<0.001)和上清液中蛋白质水平(78.20±6.16 vs 37.50±3.31,q=16.83,P<0.001)均显著升高,而敲减MCTS1则可抑制CXCL1 mRNA(0.53±0.03 vs 1.00±0.08,q=7.64,P<0.01)和蛋白质水平(17.33±1.96 vs 37.50±3.31,q=8.34,P<0.01);此外,与对照组比较,MCTS1过表达组JAK2蛋白的表达水平(2.11±0.15 vs 1.00±0.05,q=19.48,P<0.001)和STAT1的活化水平(2.10±0.19 vs 1.00±0.10,q=15.02,P<0.001)显著升高,而MCTS1敲减组JAK2蛋白水平(0.56±0.06 vs 1.00±0.05,q=7.61,P<0.01)和STAT1的活化水平均显著降低(0.46±0.07 vs 1.00±0.05,q=7.45,P<0.01)。结论肺腺癌细胞MCTS1通过JAK2-STAT1信号通路调控CXCL1的表达,并介导MDSCs的趋化。展开更多
Diabetic retinopathy is a prominent cause of blindness in adults,with early retinal ganglion cell loss contributing to visual dysfunction or blindness.In the brain,defects inγ-aminobutyric acid synaptic transmission ...Diabetic retinopathy is a prominent cause of blindness in adults,with early retinal ganglion cell loss contributing to visual dysfunction or blindness.In the brain,defects inγ-aminobutyric acid synaptic transmission are associated with pathophysiological and neurodegenerative disorders,whereas glucagon-like peptide-1 has demonstrated neuroprotective effects.However,it is not yet clear whether diabetes causes alterations in inhibitory input to retinal ganglion cells and whether and how glucagon-like peptide-1 protects against neurodegeneration in the diabetic retina through regulating inhibitory synaptic transmission to retinal ganglion cells.In the present study,we used the patch-clamp technique to recordγ-aminobutyric acid subtype A receptor-mediated miniature inhibitory postsynaptic currents in retinal ganglion cells from streptozotocin-induced diabetes model rats.We found that early diabetes(4 weeks of hyperglycemia)decreased the frequency of GABAergic miniature inhibitory postsynaptic currents in retinal ganglion cells without altering their amplitude,suggesting a reduction in the spontaneous release ofγ-aminobutyric acid to retinal ganglion cells.Topical administration of glucagon-like peptide-1 eyedrops over a period of 2 weeks effectively countered the hyperglycemia-induced downregulation of GABAergic mIPSC frequency,subsequently enhancing the survival of retinal ganglion cells.Concurrently,the protective effects of glucagon-like peptide-1 on retinal ganglion cells in diabetic rats were eliminated by topical administration of exendin-9-39,a specific glucagon-like peptide-1 receptor antagonist,or SR95531,a specific antagonist of theγ-aminobutyric acid subtype A receptor.Furthermore,extracellular perfusion of glucagon-like peptide-1 was found to elevate the frequencies of GABAergic miniature inhibitory postsynaptic currents in both ON-and OFF-type retinal ganglion cells.This elevation was shown to be mediated by activation of the phosphatidylinositol-phospholipase C/inositol 1,4,5-trisphosphate receptor/Ca2+/protein kinase C signaling pathway downstream of glucagon-like peptide-1 receptor activation.Moreover,multielectrode array recordings revealed that glucagon-like peptide-1 functionally augmented the photoresponses of ON-type retinal ganglion cells.Optomotor response tests demonstrated that diabetic rats exhibited reductions in visual acuity and contrast sensitivity that were significantly ameliorated by topical administration of glucagon-like peptide-1.These results suggest that glucagon-like peptide-1 facilitates the release ofγ-aminobutyric acid onto retinal ganglion cells through the activation of glucagon-like peptide-1 receptor,leading to the de-excitation of retinal ganglion cell circuits and the inhibition of excitotoxic processes associated with diabetic retinopathy.Collectively,our findings indicate that theγ-aminobutyric acid system has potential as a therapeutic target for mitigating early-stage diabetic retinopathy.Furthermore,the topical administration of glucagon-like peptide-1 eyedrops represents a non-invasive and effective treatment approach for managing early-stage diabetic retinopathy.展开更多
文摘目的探讨肺腺癌细胞恶性T细胞扩增序列1(MCTS1)介导骨髓源性抑制细胞(MDSCs)趋化的作用机制。方法利用生物信息学(http://timer.cistrome.org/#tab-8258-2)分析癌症基因组图谱(TCGA)中肺腺癌组织(n=515)MCTS1的表达水平与MDSCs浸润之间的相关性;利用IL-6+粒细胞-巨噬细胞集落刺激因子(GM-CSF)共同诱导人外周血单个核细胞(PBMCs)中MDSCs的生成,流式细胞术检测MDSCs生成比例,CD33磁珠分选MDSCs;收集肺腺癌SPC-A1细胞野生型以及MCTS1敲减和过表达SPC-A1细胞的无血清细胞上清液,利用Transwell小室进行MDSCs的趋化试验并计算趋化指数;羧基荧光素二醋酸盐琥珀酰亚胺酯(CFSE)标记CD8^(+)T细胞与发生趋化的MDSCs细胞共培养,流式细胞术检测CD8^(+)T细胞的增殖水平;实时荧光定量PCR(qRT-PCR)测定MDSCs相关趋化因子的mRNA水平;流式多因子芯片技术检测细胞上清液中C-X-C基序趋化因子配体1(CXCL1)的含量;Western blot检测janus激酶2(JAK2)的表达水平以及信号转导子和转录激活子1(STAT1)的活化水平。结果肺腺癌组织MCTS1与MDSCs浸润程度呈正相关(Spearmanρ=0.226,P<0.001);与空白对照组比较,IL-6和GM-CSF共同诱导PBMCs后可显著增加MDSCs的比例(4.95±1.03 vs 0.97±0.24,t=6.54,P<0.01);MCTS1过表达的SPC-A1细胞上清液对MDSCs的趋化指数较对照组显著升高(4.88±0.99 vs 2.94±0.27,q=6.29,P<0.01),而经MCTS1敲减的细胞上清液可使MDSCs的趋化指数显著降低(1.50±0.17 vs 2.94±0.27,q=4.69,P<0.05);MCTS1过表达的SPC-A1细胞中CXCL1 mRNA(2.79±0.16 vs 1.00±0.08,q=28.94,P<0.001)和上清液中蛋白质水平(78.20±6.16 vs 37.50±3.31,q=16.83,P<0.001)均显著升高,而敲减MCTS1则可抑制CXCL1 mRNA(0.53±0.03 vs 1.00±0.08,q=7.64,P<0.01)和蛋白质水平(17.33±1.96 vs 37.50±3.31,q=8.34,P<0.01);此外,与对照组比较,MCTS1过表达组JAK2蛋白的表达水平(2.11±0.15 vs 1.00±0.05,q=19.48,P<0.001)和STAT1的活化水平(2.10±0.19 vs 1.00±0.10,q=15.02,P<0.001)显著升高,而MCTS1敲减组JAK2蛋白水平(0.56±0.06 vs 1.00±0.05,q=7.61,P<0.01)和STAT1的活化水平均显著降低(0.46±0.07 vs 1.00±0.05,q=7.45,P<0.01)。结论肺腺癌细胞MCTS1通过JAK2-STAT1信号通路调控CXCL1的表达,并介导MDSCs的趋化。
基金supported by the National Natural Science Foundation of China,Nos.32070989(to YMZ),31872766(to YMZ),81790640(to XLY),and 82070993(to SJW)the grant from Sanming Project of Medicine in Shenzhen,No.SZSM202011015(to XLY)。
文摘Diabetic retinopathy is a prominent cause of blindness in adults,with early retinal ganglion cell loss contributing to visual dysfunction or blindness.In the brain,defects inγ-aminobutyric acid synaptic transmission are associated with pathophysiological and neurodegenerative disorders,whereas glucagon-like peptide-1 has demonstrated neuroprotective effects.However,it is not yet clear whether diabetes causes alterations in inhibitory input to retinal ganglion cells and whether and how glucagon-like peptide-1 protects against neurodegeneration in the diabetic retina through regulating inhibitory synaptic transmission to retinal ganglion cells.In the present study,we used the patch-clamp technique to recordγ-aminobutyric acid subtype A receptor-mediated miniature inhibitory postsynaptic currents in retinal ganglion cells from streptozotocin-induced diabetes model rats.We found that early diabetes(4 weeks of hyperglycemia)decreased the frequency of GABAergic miniature inhibitory postsynaptic currents in retinal ganglion cells without altering their amplitude,suggesting a reduction in the spontaneous release ofγ-aminobutyric acid to retinal ganglion cells.Topical administration of glucagon-like peptide-1 eyedrops over a period of 2 weeks effectively countered the hyperglycemia-induced downregulation of GABAergic mIPSC frequency,subsequently enhancing the survival of retinal ganglion cells.Concurrently,the protective effects of glucagon-like peptide-1 on retinal ganglion cells in diabetic rats were eliminated by topical administration of exendin-9-39,a specific glucagon-like peptide-1 receptor antagonist,or SR95531,a specific antagonist of theγ-aminobutyric acid subtype A receptor.Furthermore,extracellular perfusion of glucagon-like peptide-1 was found to elevate the frequencies of GABAergic miniature inhibitory postsynaptic currents in both ON-and OFF-type retinal ganglion cells.This elevation was shown to be mediated by activation of the phosphatidylinositol-phospholipase C/inositol 1,4,5-trisphosphate receptor/Ca2+/protein kinase C signaling pathway downstream of glucagon-like peptide-1 receptor activation.Moreover,multielectrode array recordings revealed that glucagon-like peptide-1 functionally augmented the photoresponses of ON-type retinal ganglion cells.Optomotor response tests demonstrated that diabetic rats exhibited reductions in visual acuity and contrast sensitivity that were significantly ameliorated by topical administration of glucagon-like peptide-1.These results suggest that glucagon-like peptide-1 facilitates the release ofγ-aminobutyric acid onto retinal ganglion cells through the activation of glucagon-like peptide-1 receptor,leading to the de-excitation of retinal ganglion cell circuits and the inhibition of excitotoxic processes associated with diabetic retinopathy.Collectively,our findings indicate that theγ-aminobutyric acid system has potential as a therapeutic target for mitigating early-stage diabetic retinopathy.Furthermore,the topical administration of glucagon-like peptide-1 eyedrops represents a non-invasive and effective treatment approach for managing early-stage diabetic retinopathy.
