本文關(guān)鍵詞：PD-L1免疫負(fù)調(diào)節(jié)作用與延長移植物存活及其機制　出處：《華中科技大學(xué)》2009年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： I型糖尿病 NIT-1細(xì)胞 PD-L1 同種免疫應(yīng)答 移植

【摘要】： 【目的】 I型糖尿病是一種主要由T細(xì)胞介導(dǎo)的，CD4~+、CD8~+T細(xì)胞和巨噬細(xì)胞浸潤胰島導(dǎo)致分泌胰島素的β細(xì)胞受損，使胰島素分泌減少，導(dǎo)致胰島素絕對缺乏。目前已證實該疾病和自身免疫耐受缺失有關(guān)，，自身反應(yīng)性T細(xì)胞在I型糖尿病的免疫發(fā)病機理中起主導(dǎo)作用。PD-L1已被證實為免疫負(fù)性調(diào)節(jié)受體PD-1的配體，文獻(xiàn)證明PD-L1在免疫負(fù)性調(diào)節(jié)及外周耐受中發(fā)揮重要作用。本實驗構(gòu)建PD-L1表達(dá)載體，轉(zhuǎn)染NIT細(xì)胞，通過體內(nèi)外實驗探討PD-L1免疫調(diào)節(jié)作用，研究其在鏈脲佐菌素(STZ)誘導(dǎo)的糖尿病小鼠模型中延長胰島移植存活時間及其作用機制。 【方法】 1．穩(wěn)定表達(dá)pPD-L1的NIT-1細(xì)胞株的建立 用Lipofectamine~(TM)2000將pPD-L1-EGFP n1和pEGFPn1兩個質(zhì)粒分別轉(zhuǎn)染NIT-1細(xì)胞系獲得穩(wěn)定轉(zhuǎn)染細(xì)胞株，分別命名為NIT-PD-L1和NIT-EGFP。 2．致敏淋巴細(xì)胞的制備 分別用三組刺激細(xì)胞NIT-1、NIT-PD-L1和NIT-EGFP體內(nèi)腹腔注射Balb／c小鼠，間隔1周再腹腔注射一次，細(xì)胞免疫后第14天分離Balb／c小鼠脾臟單個核細(xì)胞作為致敏淋巴細(xì)胞備用。 3．混合細(xì)胞培養(yǎng) 將經(jīng)過絲裂霉素C處理的NIT-1、NIT-PD-L1和NIT-EGFP三組細(xì)胞作為刺激細(xì)胞分別與新鮮分離的脾臟淋巴細(xì)胞和(或)上述致敏淋巴細(xì)胞進(jìn)行混合培養(yǎng)7天。 4．細(xì)胞增殖實驗 用CFSE預(yù)染上述小鼠淋巴細(xì)胞，待細(xì)胞混合培養(yǎng)7天后，F(xiàn)CM檢測淋巴細(xì)胞的增殖。 5．細(xì)胞凋亡檢測 收集上述細(xì)胞混合培養(yǎng)7天后的淋巴細(xì)胞，Annexin V-cy5和PI染色，F(xiàn)CM檢測淋巴細(xì)胞的凋亡。 6．細(xì)胞毒實驗 分別用三組刺激細(xì)胞NIT-1、NIT-PD-L1和NIT-EGFP體內(nèi)腹腔注射Balb／c小鼠，間隔1周再腹腔注射一次，細(xì)胞免疫后第14天分離小鼠脾淋巴細(xì)胞，將刺激細(xì)胞與上述獲得的脾淋巴細(xì)胞以1：10的比例混合培養(yǎng)，3天后收集淋巴細(xì)胞作為效應(yīng)細(xì)胞。將NIT-1細(xì)胞用CFSE染色，作為靶細(xì)胞，并以1：20的比例與效應(yīng)細(xì)胞混合培養(yǎng)，4小時后用PI染色，F(xiàn)CM檢測壞死細(xì)胞數(shù)。 7．細(xì)胞因子檢測 收集上述混合細(xì)胞培養(yǎng)上清，ELISA檢測細(xì)胞因子的分泌水平；收集上述混合培養(yǎng)后的淋巴細(xì)胞，F(xiàn)CM檢測淋巴細(xì)胞內(nèi)細(xì)胞因子的表達(dá)。 8．PD-L1誘導(dǎo)同種胰島移植耐受實驗 (1)STZ誘導(dǎo)糖尿病模型 用小劑量多次腹腔注射STZ的方法誘導(dǎo)糖尿病模型。 (2)細(xì)胞腹腔移植 將NIT-1、NIT-EGFP或NIT-PD-L1細(xì)胞(1.5×10~7)注射到Balb／c糖尿病模型小鼠腹腔。 (3)血糖、體重、胰島素釋放實驗以及生存時間觀察 移植后每隔一天監(jiān)測一次血糖、體重及生存時間；在移植后第7天和24天，用放免法檢測葡萄糖刺激的胰島素釋放量實驗。 (4)細(xì)胞增殖、細(xì)胞凋亡、培養(yǎng)上清細(xì)胞因子及胞內(nèi)細(xì)胞因子的測定方法同上 (5)小鼠血清細(xì)胞因子測定 移植后第7天，收集小鼠血清，ELISA檢測細(xì)胞因子的表達(dá)。 (6)腹腔淋巴細(xì)胞凋亡以及腹腔沖洗細(xì)胞免疫熒光染色 移植后第7天，收集小鼠腹腔沖洗細(xì)胞，F(xiàn)CM檢測腹腔淋巴細(xì)胞凋亡；免疫熒光染色檢測移植細(xì)胞的排斥情況。 【結(jié)果】 1．PD-L1對同種淋巴細(xì)胞的調(diào)節(jié)作用 (1)PD-L1對同種淋巴細(xì)胞的增殖與凋亡的影響：新鮮分離的小鼠脾臟淋巴細(xì)胞作為反應(yīng)細(xì)胞，與未修飾的NIT-1或PD-L1修飾的NIT-1(NIT-PD-L1)作為刺激細(xì)胞混合培養(yǎng)，F(xiàn)CM檢測結(jié)果表明未修飾的NIT-1刺激的淋巴細(xì)胞增殖率顯著高于NIT-PD-L1刺激的增殖率； 未修飾的NIT-1致敏淋巴細(xì)胞作為反應(yīng)細(xì)胞，與未修飾的NIT-1或NIT-PD-L1作為刺激細(xì)胞混合培養(yǎng)，F(xiàn)CM檢測結(jié)果表明未修飾的NIT-1刺激的淋巴細(xì)胞增殖率顯著高于NIT-PD-L1刺激的增殖率，而NIT-1誘導(dǎo)的淋巴細(xì)胞凋亡率低于NIT-PD-L1誘導(dǎo)的淋巴細(xì)胞凋亡率； NIT-PD-L1致敏的淋巴細(xì)胞作為反應(yīng)細(xì)胞，與未修飾的NIT-1或NIT-PD-L1作為刺激細(xì)胞混合培養(yǎng)，NIT-PD-L1刺激的淋巴細(xì)胞增殖率顯著低于未修飾的NIT刺激的淋巴細(xì)胞增殖，且可誘導(dǎo)反應(yīng)細(xì)胞凋亡。結(jié)果表明PD-L1可抑制新鮮分離的初次反應(yīng)性淋巴細(xì)胞和致敏淋巴細(xì)胞的增殖，并促進(jìn)其凋亡。 (2)PD-L1對細(xì)胞因子的表達(dá)與分泌的影響：FCM檢測淋巴細(xì)胞內(nèi)細(xì)胞因子以及ELISA檢測分泌性細(xì)胞因子結(jié)果表明，與NIT-1或NIT-EGFP誘導(dǎo)的淋巴細(xì)胞相比，NIT-PD-L1細(xì)胞誘導(dǎo)的淋巴細(xì)胞表達(dá)與分泌IL-4、IL-10水平顯著增高，而IFN-γ水平明顯降低。 (3)PD-L1對CTL活性的影響：通過NIT-1、NIT-PD-L1或NIT-EGFP三組細(xì)胞分別體內(nèi)外聯(lián)合刺激獲得的脾淋巴細(xì)胞，分別與NIT-1細(xì)胞體外共培養(yǎng)后，CFSE與PI雙染色，F(xiàn)CM檢測結(jié)果表明，NIT-1或NIT-EGFP細(xì)胞誘導(dǎo)的脾淋巴細(xì)胞介導(dǎo)NIT-1靶細(xì)胞中度壞死，而NIT-PD-L1細(xì)胞誘導(dǎo)的脾淋巴細(xì)胞介導(dǎo)NIT-1靶細(xì)胞輕度壞死，結(jié)果表明PD-L1可體內(nèi)外抑制CTL的激活與效應(yīng)。 2．PD-L1延長STZ誘導(dǎo)的糖尿病小鼠胰島移植物的存活 將NIT-1、NIT-PD-L1和NIT-EGFP三組細(xì)胞分別移植入STZ誘導(dǎo)的Balb／c糖尿病小鼠腹腔，觀察移植前后的血糖、體重、糖刺激的胰島素釋放及生存時間，以及脾臟細(xì)胞的增殖與凋亡、細(xì)胞因子的表達(dá)與分泌，實驗結(jié)果表明： (1)PD-L1對糖尿病小鼠體重、血糖及存活時間的影響：移植NIT-1、NIT-PD-L1或NIT-EGFP細(xì)胞后第3天，各組糖尿病小鼠血糖均降至正常范圍，其中移植NIT-1或NIT-EGFP細(xì)胞的對照組糖尿病小鼠血糖，在第7天左右升高并超過正常血糖水平；而移植NIT-PD-L1細(xì)胞的糖尿病小鼠可維持正常血糖水平約21天，隨后逐漸升高，于移植后第24天血糖升高且超過正常上限(＞11.1mmol／L)； 在移植后第7天，移植NIT-1或NIT-EGFP細(xì)胞的對照組糖尿病小鼠體重逐漸下降，而移植NIT-PD-L1的糖尿病小鼠體重逐漸增加；移植NIT-PD-L1細(xì)胞糖尿病小鼠的生存時間比移植NIT-1或NIT-EGFP細(xì)胞的糖尿病小鼠明顯延長。 (2)葡萄糖刺激的胰島素釋放：進(jìn)一步檢測葡萄糖刺激的胰島素釋放，由于移植NIT-1或NIT-EGFP細(xì)胞的糖尿病小鼠在移植后24天內(nèi)均死亡，所以24天組僅包括移植NIT-PD-L1細(xì)胞的小鼠。檢測結(jié)果表明，在移植后第7天，移植NIT-1或NIT-EGFP細(xì)胞的小鼠胰島素釋放量明顯低于正常小鼠，而移植NIT-PD-L1細(xì)胞的小鼠胰島素釋放量與正常小鼠對照組相比無顯著性差異，表明NIT-PD-L1細(xì)胞維持正常功能；但在移植后24天，移植NIT-PD-L1細(xì)胞小鼠的胰島素釋放量低于正常對照組小鼠，提示可能發(fā)生排斥反應(yīng)。 (3)PD-L1延長移植物存活的免疫學(xué)機制：為了研究PD-L1延長移植物存活的機制，在細(xì)胞移植后第7天，分別檢測了脾淋巴細(xì)胞和腹腔淋巴細(xì)胞的增殖與凋亡。FCM檢測結(jié)果表明，移植NIT-1或NIT-EGFP細(xì)胞的小鼠脾淋巴細(xì)胞增殖反應(yīng)顯著高于NIT-PD-L1細(xì)胞移植小鼠，而細(xì)胞凋亡率低于NIT-PD-L1細(xì)胞移植的小鼠； 在移植后第7天，F(xiàn)CM檢測脾淋巴細(xì)胞內(nèi)細(xì)胞因子以及ELISA檢測細(xì)胞培養(yǎng)上清和血清細(xì)胞因子結(jié)果表明，移植NIT-1或NIT-EGFP細(xì)胞的小鼠脾淋巴細(xì)胞內(nèi)IFN-γ表達(dá)明顯高于NIT-PD-L1細(xì)胞移植小鼠，而IL-4和IL-10顯著低于移植NIT-PD-L1細(xì)胞的小鼠；脾淋巴細(xì)胞培養(yǎng)上清與小鼠血清中細(xì)胞因子檢測結(jié)果與胞內(nèi)細(xì)胞因子的表達(dá)類似。上述結(jié)果表明PD-L1抑制淋巴細(xì)胞增殖同時促進(jìn)其凋亡；胞內(nèi)細(xì)胞因子和ELISA檢測結(jié)果顯示，PD-L1抑制IFN-γ的表達(dá)，而增加IL-4和IL-10的產(chǎn)生，從而促進(jìn)Th1細(xì)胞向Th2細(xì)胞漂移。 (4)腹腔沖洗細(xì)胞活性：腹腔沖洗細(xì)胞免疫熒光染色結(jié)果顯示，移植NIT-PD-L1細(xì)胞的小鼠腹腔中胰島素陽性細(xì)胞數(shù)量明顯高于移植NIT-1或NIT-EGFP細(xì)胞的小鼠，而移植NIT-PD-L1細(xì)胞小鼠腹腔中淋巴細(xì)胞凋亡數(shù)增高，且浸潤程度顯著低于移植NIT-1或NIT-EGFP細(xì)胞的小鼠組。 【結(jié)論】PD-L1基因修飾的NIT可明顯抑制同種淋巴細(xì)胞的增殖反應(yīng)，同時誘導(dǎo)其凋亡；PD-L1可抑制CTL的活性，且可誘導(dǎo)Th2細(xì)胞因子的分泌，抑制Th1細(xì)胞因子的分泌；從而移植延長糖尿病小鼠移植物存活時間，并維持較長的正常血糖水平，研究結(jié)果為進(jìn)一步通過誘導(dǎo)免疫耐受重建IDDM胰島細(xì)胞功能研究奠定了基礎(chǔ)。
[Abstract]:[Objective]
Type I diabetes is a mainly mediated by T cells, CD4~+, leading to insulin secreting beta cell damage in CD8~+T cells and macrophage infiltration in pancreatic islets, the decrease of insulin secretion, leading to absolute insulin deficiency. It has been confirmed that the disease and the deficiency of the immune tolerance, autoreactive T cells play a dominant role of.PD-L1 has been confirmed as a negative regulator of immune ligand receptor PD-1 in the pathogenesis of type I diabetes in the literature that PD-L1 play an important role in regulating the immune negative and peripheral tolerance. The constructed PD-L1 expression vector and transfected into NIT cells in vitro and in vivo study of PD-L1 immunomodulatory effects on the STZ prime (STZ) to extend the survival time of islet transplantation and its mechanism of diabetic mice induced.
[method]
The establishment of 1. stable NIT-1 cell line expressing pPD-L1.
Lipofectamine~ (TM) 2000 pPD-L1-EGFP N1 and two pEGFPn1 plasmids were transfected into NIT-1 cell lines stably transfected cell line, named NIT-PD-L1 and NIT-EGFP.
2. preparation of sensitized lymphocytes
With three group cells stimulated NIT-1, NIT-PD-L1 and NIT-EGFP in vivo by intraperitoneal injection of Balb / c mice, 1 weeks apart with a single intraperitoneal injection of immune cells, fourteenth days after the separation of Balb / c mice spleen mononuclear cells of sensitized lymphocytes as spare.
3. mixed cell culture
Pretreated with mitomycin C for NIT-1, NIT-PD-L1 and NIT-EGFP three cells as spleen and lymphocyte stimulating cells were freshly isolated and (or) the sensitized lymphocytes were co cultured for 7 days.
4. cell proliferation experiment
With the CFSE pre stained mouse lymphocytes, cells to be mixed after 7 days of culture, FCM lymphocyte proliferation.
5. detection of apoptosis
Collecting the cells after 7 days of culture mixed lymphocyte, Annexin V-cy5 and PI staining, detection of apoptosis of FCM lymphocytes.
6. cytotoxicity test
With three group cells stimulated NIT-1, NIT-PD-L1 and NIT-EGFP in vivo by intraperitoneal injection of Balb / c mice, 1 weeks apart with a single intraperitoneal injection of mouse spleen lymphocyte separation, fourteenth days after the stimulation of cellular immunity, cell and the spleen lymphocyte mixed by 1:10 culture, collected 3 days after NIT-1 lymphocytes were used as effector cells. Cells with CFSE staining, as target cells, and to the ratio of 1:20 cells and the effect of mixed culture, 4 hours after PI staining, cell necrosis detection FCM number.
Detection of 7. cytokines
Collect the mixed cell culture supernatant, the secretion level of ELISA cytokine detection; the collection of lymphocytes after mixed culture, the expression of cytokines in FCM lymphocytes in the detection.
Islet allograft tolerance test induced by 8.PD-L1
(1) diabetes model induced by STZ
Induction of diabetic model by low dose repeated intraperitoneal injection of STZ.
(2) cells in peritoneal transplantation
NIT-1, NIT-EGFP or NIT-PD-L1 cells (1.5 * 10~7) was injected into Balb / C diabetic mouse model of peritoneal cavity.
(3) the blood glucose, body weight, insulin release test and survival time were observed
Every day after transplantation to monitor a blood glucose, body weight and survival time; in seventh days and 24 days after transplantation, detected by radioimmunoassay of glucose stimulated insulin release test.
(4) cell proliferation, apoptosis, cytokine secretion culture assay and intracellular cytokine.
(5) determination of mouse serum cell factor
Seventh days after transplantation, serum were collected, to detect the expression of ELISA cytokines.
(6) apoptosis of peritoneal lymphocytes and peritoneal lavage cells by immunofluorescence staining
Seventh days after transplantation, mice peritoneal lavage cells, peritoneal lymphocyte apoptosis detection FCM; rejection of the transplanted cells by immunofluorescence staining.
[results]
The effect of 1.PD-L1 on regulation of allogeneic lymphocytes
(1) effect of PD-L1 on proliferation and apoptosis of allogeneic lymphocytes: freshly isolated mouse spleen lymphocytes as reaction cells with NIT-1 or PD-L1 modified NIT-1 (NIT-PD-L1) as stimulator cells in mixed culture, FCM test results show that the unmodified NIT-1 stimulated lymphocyte proliferation rate was significantly higher than that of NIT-PD-L1 stimulated proliferation rate;
Unmodified NIT-1 sensitized lymphocytes as the reaction cell, and NIT-1 or NIT-PD-L1 as stimulator cells in mixed culture, FCM test results show that the unmodified NIT-1 stimulated lymphocyte proliferation rate was significantly higher than that of NIT-PD-L1 stimulated proliferation rate and lymph cell apoptosis induced by NIT-1 was lower than the rate of lymphocyte apoptosis induced by NIT-PD-L1;
NIT-PD-L1 sensitized lymphocytes as reaction cells, and NIT-1 or NIT-PD-L1 as stimulator cells mixed culture was significantly lower than that of unmodified NIT stimulated lymphocyte proliferation rate NIT-PD-L1 stimulated lymphocyte proliferation and induce apoptosis of lymphocyte reaction. The results show that the initial reaction of PD-L1 can inhibit the freshly isolated and sensitized lymphocytes proliferation, and promote their apoptosis.
(2) effect on the expression of PD-L1 and secretion of cytokines: cytokines of FCM lymphocytes in the detection and ELISA detection of cytokines secretion results show that compared with NIT-1 or NIT-EGFP induced lymphocyte NIT-PD-L1 cell induced lymphocyte expression and secretion of IL-4, IL-10 levels were significantly higher, while IFN- levels decreased significantly.
(3) the effect of PD-L1 on the activity of CTL by NIT-1, NIT-PD-L1 or NIT-EGFP three group cells were obtained in vitro and in vivo combined stimulation of spleen lymphocytes were co cultured with NIT-1 cells in vitro, CFSE and PI double staining, FCM assay showed that the lymphocyte mediated NIT-1 or NIT-EGFP cells induced by moderate necrosis of NIT-1 cells however, spleen lymphocyte mediated NIT-PD-L1 cells induced by mild necrosis of NIT-1 cells, and the results show that the activation effect of PD-L1 and cortisol suppression of CTL.
The survival of 2.PD-L1 prolonged islet graft induced by STZ
NIT-1, Balb / C diabetic mice NIT-PD-L1 and NIT-EGFP three groups of cells were transplanted into STZ induced observation before and after transplantation, blood glucose, body weight, insulin release and survival time of glucose stimulation, and the proliferation and apoptosis of spleen cells, expression and secretion of cytokines, the experimental results show that:
(1) PD-L1 on diabetic mice weight, influence and survival time of blood glucose: the transplantation of NIT-1, NIT-PD-L1 or NIT-EGFP cells after third days, the blood glucose of diabetic mice were decreased to normal range, the transplantation of NIT-1 or NIT-EGFP cells in control group blood glucose in diabetic mice, in about seventh days and increased more than the normal blood glucose level and maintain normal blood glucose; the level of about 21 days can be transplanted NIT-PD-L1 cells in diabetic mice, then gradually increased. In twenty-fourth days after transplantation and increased blood glucose than the upper limit of normal (11.1mmol / L);
On the seventh day after transplantation, transplantation of NIT-1 or NIT-EGFP cells in the control group of diabetic mice weight decreased gradually, and the diabetic mice transplanted NIT-PD-L1 weight increased gradually; the transplanted NIT-PD-L1 cells in diabetic mice survival time was longer than NIT-1 or NIT-EGFP cells transplantation in diabetic mice.
(2) glucose stimulated insulin release: further detection of glucose stimulated insulin release, the transplantation of NIT-1 or NIT-EGFP cells in diabetic mice were killed in 24 days after transplantation, so the 24 day group includes only the transplantation of NIT-PD-L1 cells in mice. The detection results show that in the seventh days after transplantation, transplantation of insulin was significantly lower than that in normal mice NIT-1 or NIT-EGFP cells and mouse insulin release, transplantation of NIT-PD-L1 cells release and normal mice had no significant difference compared to control group, indicating that NIT-PD-L1 cells maintain normal function; but on the 24 day after transplantation, mice transplanted NIT-PD-L1 cells insulin release is lower than that of normal control mice, suggesting that the occurrence of rejection.
(3) PD-L1 extended shift immunological mechanism of plant survival: in order to study the extension of the PD-L1 shift mechanism of plant survival, in cells seventh days after transplantation, respectively to detect.FCM proliferation and apoptosis detection results of splenic lymphocytes and peritoneal lymphocytes showed that the proliferation of splenic lymphocytes of mice transplanted with NIT-1 or NIT-EGFP cells was significantly higher than that of mouse NIT-PD-L1 cells. The apoptosis rate is lower than the NIT-PD-L1 cell transplantation in mice;
On the seventh day after transplantation, cytokine FCM detection and ELISA detection in spleen lymphocytes in cell culture supernatants and serum cytokine results showed that the expression of mouse NIT-PD-L1 cells was significantly higher than that of IFN- gamma spleen lymphocytes of mice transplanted with NIT-1 or NIT-EGFP cells, while IL-4 and IL-10 were significantly lower than the transplantation of NIT-PD-L1 cells of mice spleen lymphocyte culture; cytokine expression the detection results of cytokines in serum and supernatant and intracellular similar. These results indicated that PD-L1 inhibited lymphocyte proliferation and promote apoptosis; results of cytokines and ELISA cells showed that PD-L1 inhibits expression of IFN- gamma, increased IL-4 and IL-10 production, thus promoting Th1 cell to Th2 cell drift.
(4) peritoneal cell activity: peritoneal cell immunofluorescence staining showed that the number of insulin positive cells in mice by intraperitoneal transplantation of NIT-PD-L1 cells was significantly higher than that in the transplantation of NIT-1 or NIT-EGFP cells in mice, and transplanted NIT-PD-L1 cells in mouse peritoneal lymphocyte apoptosis increased, and the degree of infiltration was significantly lower than that of mice transplanted with NIT-1 or NIT-EGFP cells.
[Conclusion] the proliferative response of PD-L1 gene modified NIT can inhibit allogeneic lymphocytes, and induce apoptosis; PD-L1 can inhibit CTL activity and induce Th2 secretion, cytokine secretion, inhibit Th1 cell factor; and diabetic mice transplantation prolongs the survival time of the grafts to normal blood glucose levels and long, research the results for further laid the foundation for the study of IDDM function of islet cell immune tolerance induced by reconstruction.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R587.1;R392

【引證文獻(xiàn)】

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1 李濤;泡球蚴感染誘導(dǎo)Kupfter細(xì)胞表達(dá)PD-L1抑制大鼠肝移植急性排斥反應(yīng)的機制研究[D];新疆醫(yī)科大學(xué);2012年

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