在线观看高清无码国产精品_狠狠噜☆天天噜日日噜av_国产激情视频六区_亚州精品视频无码精品无码_波多野结衣中字av专区在线观看_中文自拍欧美影视_18+美女黄色网站_欧美一级黄色片免费播放_国产色综合久久中文字幕av_j8又大又粗又硬之三个闺蜜

    利用層浪FongCyte?進(jìn)行流式細(xì)胞術(shù)檢測(cè)，分析在不同濃度GaS?處理后細(xì)胞中BCECF的熒光信號(hào)變化，并加以免疫熒光實(shí)驗(yàn)的驗(yàn)證，證明GaS_x能被細(xì)胞有效攝取并分解為有效成分。其次通過(guò)Western Blot檢測(cè)TfR1-FPN1的蛋白含量實(shí)驗(yàn)，ICP-MS定量分析細(xì)胞內(nèi)鐵含量實(shí)驗(yàn)和轉(zhuǎn)錄組測(cè)序?qū)嶒?yàn)，進(jìn)一步驗(yàn)證GaS?具有干擾鐵代謝的潛力。

Figure 2. GaSx disrupted tumor cell iron metabolism. (a)Confocal images of CT26 cell endocytosis after GaSx incubation for different time. (b)Confocal images of WSP-1 fuorescence staining of CT26 cells after diferent treatments. (c)BECEF signaling detection in CT26 cells by flow cytometry after treatment with different concentrations of GaSx. (d)Statistical analysis of the BECEF signals in each group in (b). (e)WB detection of iron metabolism regulatory axis-related proteins, including TfR1 and FPN1, in CT26 cells after different treatments. (f)Determination of Ga and Fe contents in CT26 cells after GaSx treatment for different time by ICP-MS. (g)Clustering heat map of differential genes related to iron metabolism. (h)Scheme of the mechanism by which GaSx disrupted iron metabolism in tumor cells.

GaS?干擾鐵代謝導(dǎo)致線粒體膜電位喪失、功能障礙，促使Bax表達(dá)上調(diào)、細(xì)胞色素 c(Cyt C)釋放，激活caspase-3依賴的凋亡途徑，同時(shí)誘導(dǎo)Endo G和AIF等釋放，觸發(fā)caspase-3非依賴的凋亡途徑，造成 DNA 損傷。GaS?對(duì)腫瘤細(xì)胞有濃度依賴性細(xì)胞毒性，對(duì)正常細(xì)胞毒性低，凋亡抑制劑可降低其細(xì)胞毒性。作者通過(guò)Annexin V - FITC 檢測(cè)，使用層浪FongCyte?進(jìn)行數(shù)據(jù)采集，結(jié)果顯示經(jīng)硫氫化鈉和Ga(NO₃)₃處理后，凋亡細(xì)胞數(shù)量均有所增加，但與對(duì)照組相比，GaS?處理導(dǎo)致凋亡細(xì)胞數(shù)量顯著增加，證實(shí)GaS?誘導(dǎo)細(xì)胞凋亡能力強(qiáng)。

Figure 3. GaSx induced apoptosis in tumor cells. (j) Flow cytometric analysis of CT26 cell apoptosis after different treatments. (k) Scheme of the mechanism of GaSx killing tumor cells.

GaS?誘導(dǎo)類凋亡促使免疫相關(guān)細(xì)胞變化，如導(dǎo)致核蛋白高遷移率族蛋白 B1(HMGB1) 從細(xì)胞核釋放，鈣網(wǎng)蛋白（CRT）從內(nèi)質(zhì)網(wǎng)腔轉(zhuǎn)移到細(xì)胞表面，二者作為危險(xiǎn)信號(hào)啟動(dòng)炎癥反應(yīng)和免疫激活。通過(guò)檢測(cè)發(fā)現(xiàn)，GaS?處理后 CT26 細(xì)胞中HMGB1在細(xì)胞核的綠色熒光信號(hào)呈劑量依賴性減少，CRT在細(xì)胞膜的綠色熒光信號(hào)劑量依賴性增加，表明GaS?加速了損傷相關(guān)分子模式（DAMPs）釋放，增強(qiáng)了免疫反應(yīng)。DC細(xì)胞在DAMPs刺激下成熟，通過(guò)層浪FongCyte?檢測(cè)CD80和CD86陽(yáng)性比例，發(fā)現(xiàn)與GaS?處理細(xì)胞上清孵育后，成熟DCs比例以濃度依賴方式增加，表明GaS?可激活免疫反應(yīng)，通過(guò)誘導(dǎo)類凋亡發(fā)揮作用，具有克服凋亡抵抗和刺激免疫反應(yīng)的優(yōu)勢(shì)，有望與化療和免疫療法協(xié)同作用。

Figure 4. GaSx induced paraptosis in tumor cells. (j-k) Proportion of mature DCs after incubating DCs and supernatants from CT26 cells treated with different concentrations of GaSx. (l) Scheme of GaSx-induced paraptosis in tumor cells to activate the immune response.

 由于GaS?在體外具有破壞鐵代謝和誘導(dǎo)細(xì)胞凋亡的能力，作者進(jìn)一步驗(yàn)證了GaS?的體內(nèi)抗腫瘤作用。結(jié)果顯示GaS?顯著抑制腫瘤生長(zhǎng)，延長(zhǎng)小鼠中位生存期，且對(duì)小鼠體重和主要器官無(wú)明顯影響，組織學(xué)分析證實(shí)其腫瘤殺傷效果，免疫組化和免疫熒光染色表明其可調(diào)節(jié)腫瘤細(xì)胞鐵代謝相關(guān)蛋白表達(dá)。

    GaS?可誘導(dǎo)腫瘤細(xì)胞釋放DAMPs，促進(jìn)DC細(xì)胞成熟，增強(qiáng)CD4?和CD8?T細(xì)胞浸潤(rùn)，使腫瘤相關(guān)巨噬細(xì)胞表型由抑制M2表型向炎癥M1表型轉(zhuǎn)變，減少調(diào)節(jié)性T細(xì)胞（Tregs）和髓源性抑制細(xì)胞（MDSCs），增加促炎細(xì)胞因子如白細(xì)胞介素-6（IL-6）、白細(xì)胞介素-12p70（IL-12p70）和腫瘤壞死因子（TNF-α），與αPD-1聯(lián)合使用可顯著抑制腫瘤進(jìn)展，增強(qiáng)抗腫瘤免疫反應(yīng)。這些結(jié)果證實(shí)了GaS?在提高臨床免疫治療療效方面的潛力。一方面，GaS?破壞鐵代謝，觸發(fā)異常分泌和DAMPs釋放。另一方面，它通過(guò)改變瘤內(nèi)鐵分布誘導(dǎo)巨噬細(xì)胞M1極化，并通過(guò)H2S介導(dǎo)的信號(hào)調(diào)控抑制MDSCs和treg的浸潤(rùn)。這種協(xié)同的方法顯著增強(qiáng)腫瘤的免疫原性，逆轉(zhuǎn)免疫抑制的腫瘤微環(huán)境，有效地克服了免疫治療的障礙。

Figure 5. Immunological evaluation of tumors from GaSx-treated mice and the combination therapy with GaSxandαPD-1 antibody. (a)Scheme of the immuno-evaluation of tumors and TDLNs after different treatments (G1:Control, G2:NaHS, G3:Ga(NO₃)₃, and G4:GaSx). (b)Mature DCs in tumors after different treatments in (a). (c)CD8+T cells infitration in tumors after different treatments in (a). (d)CD4+T cells infitration in mouse tumors after various treatments in (a). (e)Proportion of M2 cells intumors after various treatments in (a). (f)Proportion of M1 cells in mouse tumors after various treatments in (a). (g)Tregs infitration in mouse tumors after various treatments in (a). (h)Proportion of MDSCs in mouse tumors after various treatments in (a). (i)Mature DCs in TDLNs after different treatments in (d). (j?l)Pro-infammatory cytokine levels (j) IL-6, (k) IL-12p70, and (l) TNF-αin tumors from the mice after various treatments in (a). (m?o)Pro-infammatory cytokine levels (m) IL-6, (n) IL-12p70, and (o) TNF-αin the TDLNs of the mice after various treatments are shown in (a). (p)Scheme ofGaSx andαPD-1 combination therapy. (q)Tumor volume of the mice in each group (n=5) in (p). (r)Tumor weights ofthe mice after 14 days of different treatments in (p). (s)Photographs of tumors after 14 days of different treatments in (p). (t)Tumorvolume of mice after different treatments in (p). (u)H&E images and CD4⁺CD8⁺ stained tumors after 14 days of different treatments in (p).