Nanoparticles for targeted delivery of therapeutics and small interfering RNAs in hepatocellular carcinoma

Hepatocellular carcinoma(HCC) is the 5th most common malignancy which is responsible for more than half million annual mortalities; also, it is the third leading cause of cancer related death. Unfavorablesystemic side-effects of chemotherapeutic agents and susceptibility to the degradation of small interfering RNAs(si RNAs), which can knock down a specific gene involved in the disease, have hampered their clinical application. So, it could be beneficial to develop an efficient carrier for the stabilization and specific delivery of drugs and si RNA to cells. Targeted nanoparticles have gained considerable attention as an efficient drug and gene delivery system, which is due to their capability in achieving the highest accumulation of cytotoxic agents in tumor tissue, modifiable drug pharmacokinetic- and bio-distribution, improved effectiveness of treatment, and limited sideeffects. Recent studies have shed more light on the advantages of novel drug loaded carrier systems vs free drugs. Most of the animal studies have reported improvement in treatment efficacy and survival rate using novel carrier systems. Targeted delivery may be achieved passively or actively. In passive targeting, no ligand as homing device is used, while targeting is achieved by incorporating the therapeutic agent into a macromolecule or nanoparticle that passively reaches the target organ. However, in active targeting, the therapeutic agent or carrier system is conjugated to a tissue or cell-specific receptor which is overexpressed in a special malignancy using a ligand called a homing device. This review covers a broad spectrum of targeted nanoparticles as therapeutic and nonviral si RNA delivery systems, which are developed for enhanced cellular uptake and targeted gene silencing in vitro and in vivo and their characteristics and opportunities for the clinical applications of drugs and therapeutic si RNA are discussed in this article. Asialoglycoprotein receptors, low-density lipoprotein, ganglioside GM1 cell surface ligand, epidermal growth factor receptor receptors, monoclonal antibodies, retinoic acid receptors, integrin receptors targeted by Arg-Gly-Asp peptide, folate, and transferrin receptors are the most widely studied cell surface receptors which are used for the site specific delivery of drugs and si RNA-based therapeutics in HCC and discussed in detail in this article.

Therapeutic potential of small interfering RNAs/micro interfering RNA in hepatocellular carcinoma

Hepatocellular carcinoma(HCC) is the predominant form of primary liver cancer and represents the third leading cause of cancer-related death worldwide. Current available therapeutic approaches are poorly effective,especially for the advanced forms of the disease. In the last year,short double stranded RNA molecules termed small interfering RNAs(si RNAs) and micro interfering RNAs(mi RNA),emerged as interesting molecules with potential therapeutic value for HCC. The practical use of these molecules is however limited by the identification of optimal molecular targets and especially by the lack of effective and targeted HCC delivery systems. Here we focus our discussion on the most recent advances in the identification of si RNAs/mi RNAs molecular targets and on the development of suitable si RNA/mi RNAs delivery systems.

Inhibition of genes expression of SARS coronavirus by synthetic small interfering RNAs

RNA interference (RNAi) is triggered by the presence of a double-stranded RNA (dsRNA), and results in the silencing of homologous gene expression through the specific degradation of an mRNA containing the same sequence. dsRNAmediated RNAi can be used in a wide variety of eucaryotes to induce the sequence-specific inhibition of gene expression.Synthetic 21-23 nucleotide (nt) small interfering RNA (siRNA) with 2 nt 3' overhangs was recently found to mediate efficient sequence-specific mRNA degradation in mammalian cells. Here, we studied the effects of synthetic siRNA duplexes targeted to SARS coronavirus structural proteins E, M, and N in a cell culture system. Among total 26 siRNA duplexes, we obtained 3 siRNA duplexes which could sequence-specifically reduce target genes expression over 80％ at the concentration of 60 nM in Vero E6 cells. The downregulation effect was in correlation with the concentrations of the siRNA duplexes in a range of 0～60 nM. Our results also showed that many inactive siRNA duplexes may be brought to life simply by unpairing the 5' end of the antisense strands. Results suggest that siRNA is capable of inhibiting SARS coronavirus genes expression and thus may be a new therapeutic strategy for treatment of SARS.

Combination of small interfering RNAs mediates greater suppression on hepatitis B virus cccDNA in HepG2.2.15 cells

AIM： To observe the inhibition of hepatitis B virus （HBV） replication and expression in HepG2.2.15 cells by combination of small interfering RNAs （siRNAs）. METHODS： Recombinant plasmid psiI-HBV was constructed and transfected into HepG2.2.15 cells. At 48 h, 72 h and 96 h after transfection, culture media were collected and cells were harvested for HBV replication assay. HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay （ELISA）. Intracellular viral DNA and covalently closed circular DNA （cccDNA） were quantified by real-time polymerase chain reaction （PCR）. HBV viral mRNA was reverse transcribed and quantified by reverse-transcript PCR （RT-PCR）. RESULTS： siRNAs showed marked anti-HBV effects. siRNAs could specifically inhibit the expression of HBsAg and the replication of HBV DNA in a dosedependent manner. Furthermore, combination of siRNAs, compared with individual use of each siRNA, exerted a stronger inhibition on antigen expression and viral replication. More importantlycombination of siRNAs significantly suppressed HBV cccDNA amplification. CONCLUSION： Combination of siRNAs mediates a stronger inhibition on viral replication and antigenexpression in HepG2.2.15 cells, especially on cccDNA amplification.

Small interfering RNA targeting PGC-1α inhibits VEGF expression and tube formation in human retinal vascular endothelial cells

AIMTo determine whether small interfering RNA (siRNA) of PGC-1α could inhibit vascular endothelial growth factor (VEGF) expression and tube formation in human retinal vascular endothelial cells (hRVECs).METHODShRVECs transfected with peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) siRNA were incubated for 24h and then placed into a normoxic (20%, O2) or hypoxic (1%, O2) environment for another 16h. PGC-1α mRNA and protein levels were detected by real-time PCR and Western blot. VEGF mRNA and protein levels were detected by real-time PCR and ELISA. Cell proliferation was evaluated by BrdU incorporation assay. Forty-eight hours after siRNA transfection, hRVECs were planted into Matrigel-coated plates and cultured under normoxic (20%, O2) or hypoxic (1%, O2) conditions for another 48h. The tube formation of hRVECs was observed under an optical microscope and quantified by counting the number of branch points and calculating the total tube length.RESULTSPGC-1α mRNA and protein levels were significantly reduced by PGC-1α siRNA, and VEGF mRNA and protein levels also decreased significantly. The percentage of BrdU-labeled cells in siPGC-1α groups were significantly decreased compared with control siRNA groups under normoxia and hypoxia in cell proliferation assay. In the tube formation assay, PGC-1α siRNA treated cells formed significantly fewer tubes.CONCLUSIONBlocking PGC-1α expression can inhibit VEGF expression in hRVECs and inhibit their ability to form tubes under both normoxic and hypoxic conditions.

KNOCKDOWN OF SURVIVIN EXPRESSION BY SMALL INTERFERING RNA SUPPRESSES PROLIFERATION OF TWO HUMAN CANCER CELL LINES

Objective To construct an expression vector of small interfering RNA （siRNA） against survivin and observe its effects on survivin expression and proliferation of human pancreatic cancer cell line PC-2 and breast cancer cell line MCF-7. Methods Constructed an expression vector of siRNA against survivin and transfected it into PC-2 and MCF-7 cells using lipofectamine^TM 2000. The expression of survivin was detected by semi-quanfifive RT-PCR and immunohistochemistry, and its effects on proliferation of PC-2 and MCF-7 cells were detected by MTT assay. Results The introduction of sequence-specific siRNA could efficiently suppress survivin expression at both mRNA and protein levels in the two cancer cell lines. In PC-2 cells, the expression inhibition rates were 81.25% at mRNA level and 74.24% at protein level In MCF-7 cells, the expression inhibition rates were 64.91% at mRNA level and 79. 72% at protein level The proliferation of PC-2 and MCF-7 cells was also suppressed, and24 and 48 hours after the cells were reseeded, the proliferation inhibition rates of PC-2 cells were 28. 00% and 33. 38%, and that of MCF-7 cells were 31.58% and 33.02%, respectively. Conclusions The expression vector of siRNA against survivin can block survivin expression in PC-2 and MCF-7 cells efficiently and specifically. Down regulation of survivin expression can suppress proliferation of PC-2 and MCF-7 cells. Survivin RNAi may have potential value in gene therapy of human cancers.

Progress of Targeting Transforming Growth Factor-β1 Small Interfering RNA in Liver Fibrosis

Liver fibrosis is a common pathological consequence of a variety of chronic stimuli, including viral, autoimmune, drug-induced, cholestatic and metabolic diseases. Fibrosis is driven by a dynamic process involving increased synthesis of matrix components and a failure of physiological mechanisms of matrix turnover. Activation of hepatic stellate cells(HSCs) remains a central event in fibrosis. HSCs are the main source of extracellular matrix(ECM). Transforming growth factor-beta(TGF-β), which is the fibrogenic master cytokine, can induce the activation of HSCs to produce a large amount of ECM, and is capable of inducing apoptosis of liver cells. RNA interference(RNAi) is a novel gene disruption technology. Studies have shown that small interfering RNA(si RNA) targeting TGF-β1 may inhibit the activation and proliferation of HSCs, suppress ECM synthesis and block liver fibrosis. TGF-β1 si RNA-mediated gene silencing therapy provides a new avenue for liver fibrosis. This review summarizes recent progresses in research on HSCs, TGF-β1 and TGF-β1 si RNA in liver fibrosis.

Impact of Bovine Skeletal Muscle Satellite Cell Differentiation by Small Interfering RNA Targeting Myogenin Gene

To examine the effect of myogenin gene on the differentiation of bovine skeletal muscle satellite cell, we constructed small interfering RNA plasmid vector to obtain myogenin knockdown bovine skeletal muscle cells, then used cell transfection, real time RCR and Western Blot to detect the influence of myogenin to cell differentiation. Results showed that the knockdown of myogenin significantly decreased its expression and other muscle-specific genes. Compared to the control, it could differentiate into few myotubes when challenged by low serum in the medium. These findings provided an important theoretical basis for further explore of the genetic mechanism in adult skeletal muscle, the remedy of muscle injuries and the cultivation of high-yield transgenic cattle.

Effects of Small Interfering RNATargeting Sphingosine Kinase-1 Gene on the Animal Model of Alzheimer's Disease

Summary： Alzheimer＇s disease （AD） is an age-related, progressive neurodegenerative disorder that occurs gradually and results in memory, behavior, and personality changes. Abnormal sphingolipid metabolism was reported in AD previously. This study aimed to investigate whether sphK1 could exacerbate the accumulation of amyloid protein （Aβ） and sharpen the learning and memory ability of the animal model of AD using siRNA interference. An adenovirus vector expressing small interfering RNA （siRNA） against the sphK1 gene （sphKl-siRNA） was designed, and the effects of sphKl-siRNA on the APP/PS1 mouse four weeks after treatment with sphKl-siRNA hippocampal injection were examined. SphK1 protein expression was confirmed by using Western blotting and ceramide content coupled with SIP secretion was evaluated by enzyme-linked immunosorbent assay （ELISA）. Aβ load was detected by immunohistochemical staining and ELISA. Morris water maze was adopted to test the learning and memory ability of the APP/PS 1 mice. A significant difference in the expression of sphK1 protein and mRNA was observed between the siRNA group and the control group. Aβ load in transfected mice was accelerated in vivo, with significant aggravation of the learn- ing and memory ability. The sphKl gene modulation in the All load and the learning and memory ability in the animal model of AD may be important for the treatment of AD.

Inhibition of HOXB7 Gene Expression in Melanoma Cells by Small Interfering RNA

Objective： HOXB7 gene is a kind of transcription regulator over-expressed in malignant melanoma （MM） cell lines. It can specifically up-regulate the expression of angiogenic factors and tumor growth factors such as bFGF, GROa, VEGF and induce angiogenesis in melanoma, resulting in the proliferation and metastasis of tumor cells. We designed and synthesized HOXB7 specific siRNA to study its interfering effect on the expressions of HOXB7 and bFGF genes in melanoma A375 cell line and the biologic characteristics of A375 cells. Methods： Three synthesized siRNA with different sequences were separately transfected into A375 cells by lipofecter 2000. The expression of HOXB7 and bFGF mRNA in transfected cells was detected by RT-PCR 24 and 48 hours after transduction. The expression of bFGF protein in the transfected cells were detected by flowcytometry 48 hours after transfection. MTT assay was used to analyze the cell proliferation rate of siRNA transfected cells. Based on the in vitro experiment results, one effective siRNA sequence was selected for the construction of in vivo siRNA expression vector. Then, a malignant melanoma animal model was established. The siRNA expression plasmid was injected into the tumor foci and its influence on the growth and angiogenesis of tumor was observed. Results： The mRNA expressions of both HOXB7 and bFGF genes in the A375 cells reduced significantly 24 and 48 hour after transfection of siRNA. Expression level of the protein of angiogenic factor bFGF induced by HOXB7 gene in siRNA transfected cells was significantly lower than that in control cells 48 hours after transduction. Cell proliferation was also suppressed in siRNA transfected cells. Two of the three siRNA strands showed prominent interference effect. The in vivo study indicated that the tumor size and the microvessel density in the tumor both reduced after injection of HOXB7siRNA plasmid. Conclusion： Down-regulation of HOXB7 gene expression can effectively reduce the expression of angiogenic factor bFGF and the proliferation of MM cells. Besides, the growth and angiogenesis of MM tumor were also inhibited.

Silencing invariant chain of DCs enhances Th1 response using small interfering RNA

RNA interference(RNAi),which causes the degradation of any RNA in a sequence specific manner,is a posttranscriptional gene silencing mechanism.Targeting the invariant chain(Ii)in DCs has been used as an approach to enhance antitumor immunity.It is demonstrated in this article that transfection of H-2(K)DCs with siRNA specific for Ii gene can significantly knock down Ii.When exposed to TNF-alpha,immature DCs transfected with Ii siRNA can differentiate into mature DCs without reducing viability or IL-12p70 production.Ii siRNA-treated H-2(K)DCs exhibited an increased allostimulatory capacity in a lymphocyte proliferation assay.Furthermore,Ii siRNA-transfected H-2(K)DCs enhanced Th1 responses by increasing IFN-gamma and decreasing IL-4 production,and much stronger cytotoxic activity was observed when DCs were co-transfected with Ii siRNA and an endogenous tumor antigen in vitro.Our findings indicate that silencing the Ii gene in DCs with siRNA may offer a potential approach to enhancing antitumor immunotherapy.

The influence of small interfering RNA on the expression of Survivin in human glioma cells

Objective This study aims to investigate the feasibility of knockdown of Survivin gene with small interfering RNA and to observe the apoptosis in gliomas which is influenced by siRNA. Methods Survivin specific siRNA oligonucleotides were designed and synthesized artificially. This siRNA

siRNA纳米递送系统在类风湿性关节炎治疗中的研究进展

类风湿性关节炎是一种自身免疫性疾病,其病因复杂,目前尚未有很好的治疗方法,长期服用抗风湿药物也带来诸多副作用。RNA干扰技术是指通过外源性或内源性的双链RNA在体内诱导靶基因m RNA产生特异性降解,进而引起不同水平的基因沉默,具有高效、高特异性、低毒等优点,在生物医药领域有着巨大潜力。小干扰RNA(si RNA)作为RNA干扰技术的重要效应分子,在治疗类风湿关节炎方面具有巨大潜力。本文综述了siRNA递送系统用于治疗类风湿关节炎的最新研究进展,阐明了基于siRNA纳米给药系统治疗类风湿关节炎的优势,并展望了未来siRNA递送的发展方向。

Small RNA Interference-mediated Gene Silencing of TREK-1 Potassium Channel in Cultured Astrocytes

This study was aimed to examine the effect of TREK-1 silencing on the function of astrocytes. Three 21-nucleotide small interfering RNA (siRNA) duplexes (siT1, siT2, siT3) targeting TREK-1 were constructed. Cy3-labeled dsRNA oligmers were used to determine the transfection efficiency in cultured astrocytes. TREK-1-specific siRNA duplexes (siT1, siT2, siT3) at the optimal concentration were transfected into cultured astrocytes, and the most efficient siRNA was identified by the method of immunocytochemical staining and Western blotting. The proliferation of astrocytes tranfected with TREK-1-targeting siRNA under hypoxia condition was measured by fluorescence-activated cell sorting (FACS). The results showed that TREK-1 was expressed in cultured astrocytes. The dsRNA oligmers targeting TREK-1 could be transfected efficiently in cultured astrocytes and down-regulate the expression of TREK-1 in astrocytes. Moreover, the down-regulation of TREK-1 in astrocytes contributed to the proliferation of astrocytes under hypoxia condition as determined by cell cycle analysis. It was concluded that siRNA is a powerful technique that can be used to knockdown the expression of TREK-1 in astrocytes, which helps further investigate the function of TREK-1 channel in astrocytes under physicological and pathological condition.

Effect of siRNA interference on nerve growth factor in intervertebral disc inflammation rats

Objective:To investigate the inhibition effect of siRNA interference on NGF induced by inflammatory factor IL-6,and JUL—1 so as to provide novel targets for clinical treatment of discogenic low back pain.Methods:The intervertebral disc nucleus and annulus fibrosus cells of rats were separated-The cells were co-cultured with different concentrations(10 nmol/L,20nmol/L,50 nmol/L,100 nmol/L)of IL-6 and IL-1β.The NGF-siRNA was leaded into the cocultured cells with its import ability assessed by flow cytometry instrument tests,hefore and after which the NCF mRNA expression was detected by real-time Q-PCR and the NGF content was detected by ELISA.Results:Flow cytometry instrument test results showed that the NGFsiRNA cell conversion rate was 99.8%.Real-time Q-PCR detection results showed that compared with negative control group,the NGF mRNA expression of co-cultured cells treated by 10 nmol/L,20 nmol/L,50 nmol/L,100 nmol/L IL-6 and IL-1βwere respectively raised 3.4,3.7,4.7,3.7 times which were all significantly down-regulated after the import of NGF-siRNA.EILSA detection results showed that compared with negative control group,the NGF content of cocultured medium treated by 10 nmol/L,20 nmol/L,50 nmol/L,100 nmol/L I-L6 and IL-1βwere respectively raised 2.9,3.3,4.5,7.4 times which were all significantly decreased after the import of NGF-siRNA.Conclusions:These molecular biological results suggest that inflammatory factor IL-6 and IL-1βcould stimulate NCF on intervertebral disc cells in vitro culture model and its efficiency is concentration dependent,while siRNA interference can inhibit the stimulation effect of IL-6 and IL-1βon intervertebral disc cell,which provides a new targets for the clinical treatment of discogenic low back pain.

Ad-VEGF-siRNA抑制荷人骨肉瘤裸鼠血管生成的形态学研究

背景与目的:血管内皮生长因子是骨肉瘤血管形成重要的促进因子,本研究通过抗血管生成方法,观察其抑制荷人骨肉瘤裸鼠血管生成的形态学表现。方法:取4～8周龄的Balb/c裸鼠作为实验动物,人骨肉瘤细胞株MG63皮下接种法构建骨肉瘤的动物模型。建模成功后将荷人骨肉瘤的裸鼠随机分为3组,每组15只。A组使用自行构建的Ad-VEGF-siRNA干扰新生血管形成;B、C组分别使用等剂量的Ad-EGFP或PBS作为对照。实验维持19d,药物使用后隔日测量肿瘤体积和体重,绘制肿瘤生长曲线;采用常规HE染色、VEGF和CD31相关抗原免疫组化染色观察各组肿瘤标本;各组随机抽取一个标本在透射电镜下观察仿血管发生的超微结构。结果:实验结束时,A组裸鼠瘤体体积为(0.31±0.18)cm3,重量为(0.75±0.21)g,微血管密度(microvessed density,MVD)为5.79±0.34,VEGF表达为235228.09±123244.41,B、C照组裸鼠瘤体积分别为(1.21±0.29)cm3、(1.43±0.95)cm3;瘤重分别为(1.19±0.27)g、(1.19±0.35)g;MVD分别为11.00±0.68、10.42±0.10;VEGF表达分别为9641992.40±90479.62、981298.00±213590.50。A组肿瘤体积、重量、MVD和VEGF表达均显著低于B、C组。MVD和VEGF呈正相关关系,相关系数为0.9989。Ad-VEGF-siRNA组仿血管发生数量1.4000±0.5477,明显小于Ad-EGFP和PBS对照组。透射电镜可见肿瘤细胞形成的仿血管。结论:通过宏观和微观的形态学观察,证实Ad-VEGF-siRNA介导的以VEGF为靶向的抗血管生成能够抑制荷人骨肉瘤裸鼠的血管生成。

利用siRNA阻抑survivin基因表达诱导乳腺癌细胞系MCF-7细胞凋亡的研究(英文)

背景与目的:survivin属IAP基因家族成员,表达于多种肿瘤组织中,能促使细胞逃避凋亡,并促进细胞的异常有丝分裂。本研究旨在探讨敲除survivin基因后,癌细胞的增殖和凋亡情况。方法:利用RNAi阻抑人乳腺癌细胞内survivin基因的表达,RT-PCR和Westernblot法分析survivin基因mRNA和蛋白的表达,MTT法检测细胞生长增殖抑制率,流式细胞术检测细胞凋亡率。结果:survivinsiRNA转染组,survivin基因表达水平与未转染组比较下调了64%。随着siRNA浓度的增加,细胞增殖抑制率逐渐增高,200nmol/L剂量组细胞增殖抑制率最高,可达60.9%。不同浓度的siRNA可不同程度地诱导细胞凋亡,200nmol/L剂量组凋亡率最高,可达29.0%。结论:survivinsiRNA有可能成为治疗乳腺癌的新药物。

针对H1N1病毒的多特征siRNA设计

针对甲型流感病毒H1N1基因,从RNAi的角度出发,采用多特征融合的方法,进行siRNA预测。对2009年的46株病毒序列的PA片段进行分析,从经过序列分析所获得的众多靶序列中,采用结构分析手段对靶序列进行筛选,获得较易干扰的靶序列及设计出相应的siRNA。2009年爆发的H1N1病毒,序列保守性高,靶序列一致性高,结构保守性高。该方法可以有效选择可能的靶序列,并在此基础上进一步筛选,以获得少量较易干扰的靶序列,该方法为复杂序列siRNA的设计提供了新思路,对siRNA的优化设计有指导意义,有助于利用RNAi进行H1N1治疗的后续研究。

质粒介导靶向c-myc的siRNA对MCF-7乳腺癌细胞增殖的影响

目的观察靶向原癌基因c-myc的siRNA对MCF-7人乳腺癌细胞c-myc/c-Myc表达及细胞增殖的影响。方法以原癌基因c-myc mRNA 589-609位碱基为靶序列构建靶向c-myc的siRNA真核表达质粒p-Mat01-1及其错配质粒p-Mis09-1,空质粒pEGFP-C1为对照,用脂质体Lipo2000包裹转染MCF-7细胞。采用RT-PCR、Western blot检测c-myc mRNA及蛋白表达,MTT法检测MCF-7细胞增殖。结果与pEGFP-C1及p-Mis09-1比较,转染p-Mat01-1能够特异性抑制MCF-7细胞c-myc mRNA(24h:P<0.01)及蛋白(5d:P<0.01)表达,显著降低MCF-7细胞增殖能力(3d:P<0.05,5、7d:P<0.01)。结论采用siRNA表达质粒技术能够有效抑制MCF-7乳腺癌细胞c-myc/c-Myc表达,初步证实下调c-myc/c-Myc表达能够抑制MCF-7细胞增殖,为RNA干扰技术在乳腺癌生物治疗中的应用打下一定实验基础。

有效抑制小胶质细胞上TLR4表达的siRNA筛选及转染复合物细胞毒性的检测

目的筛选有效抑制小胶质细胞上TLR4(Toll-like receptor 4)表达的小干扰RNA(simall interference RNA,siR-NA)序列,并检测转染复合物的细胞毒性。方法设计并合成针对TLR4的siRNA4条(siRNA312,siRNA439,siRNA1495,siRNA2062)及1条与TLR4基因无同源性的带绿色荧光标记的阴性对照siRNA:在LipofectamineTM 2000介导下转染小胶质细胞。用RT-PCR方法测定干扰后小胶质细胞上TLR4mRNA表达,Western blot方法测定干扰后TLR4蛋白表达,比较其抑制率,筛选出有效抑制靶基因表达的siRNA。采用MTT法检测转染复合物的细胞毒性。结果①siRNA(40nmol·L-1)和LipofectamineTM 2000(1μl)有较高的转染效率;②与阴性对照组相比,TLR4 siRNA(40nmol·L-1)干扰小胶质细胞24h后TLR4 mRNA的相对表达量降低,分别为:85%(siRNA439)、73%(siRNA312)、67%(siRNA1495)和33%(siRNA2062)。③siRNA439干扰小胶质细胞48h后TLR4蛋白表达最低(P<0.01)。④LipofectamineTM 20001μl复合低于40nmol·L-1的siRNA的各组细胞存活率均在85%以上。结论①siRNA439对小胶质细胞上TLR4表达的抑制效果最大。②siRNA浓度40nmol·L-1,LipofectamineTM 2000浓度1μl,细胞毒性很低,适合转染。