细胞生物学作业.docx

1、细胞生物学作业Foe turned friend: multiple functional roles attributable to hyper-activatingstem cell factor receptor mutant in regeneration of the haematopoietic cellcompartmentS. Pati*,1, O. P. Kalra and A. Mukhopadhyay*Stem Cell Biology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, N

2、ew Delhi, India, and Department of Medicine, UniversityCollege of Medical Sciences and Guru Teg Bahadur Hospital, University of Delhi, New Delhi, IndiaReceived 1 June 2010; revision accepted 2 July 2010AbstractObjectives: Stem cell factor receptor, c-kit, isconsidered to be the master signalling mol

3、ecule ofhaematopoietic stem cells. It develops the orchestralpattern of haematopoietic cell lineages, seen by itsvarying degree of omnipresence in progenitors, lineagecommitted and mature cells. We have investigatedthe effect of over-expressing c-kit on earlyrecovery of the haematopoietic compartmen

4、t, in irradiatedhosts.Materials and methods: Normal bone marrow cells(BMCs) were transfected with Kitwt (wild-type c-kit)or its variant Kitmu (asp814tyr) by electroporation.Lethally irradiated mice were transplanted with normalor transfected congeneic BMCs. The effect ofectopic expression of c-kit o

5、n haematopoietic cellrecovery was determined by analysing donor-derivedcells. Furthermore, effects of both types of c-kit overexpressionon progenitor and lineage-committed cellswere examined by flow cytometric analysis of Sca-1and lineage-committed (Lin+) cells respectively.Results: Hyper-activating

6、 Kitmu significantly improvedrecovery of the haematopoietic system inirradiated hosts. In vivo results showed that thedonor-derived c-kit+ cell population was increased tomore than 3-fold in the case of Kitmu-transfected cellscompared to normal and Kitwt over-expressingBMCs. In general, survival of

7、progenitor andcommitted cell was improved in the Kitmuover-expressing system compared to the other twocohorts.Conclusion: These results suggest that recruitmentof the hyper-activating variant of c-kit (Kitmu) lead toearly recovery of the bone marrow of lethallyirradiated mice.IntroductionHaematopoie

8、tic stem cells (HSCs) are embodied withimmense capability to construct a complete haematopoieticcompartment from a single cell, which acts as the primitivepowerhouse (1,2). Radiation-induced damage of thebone marrow (BM) leads to myelodysplasia, resultingfrom death of progenitors, with consequent lo

9、ss of functionalcells. The current approach to successful managementof treating malignancy includes radiation and orchemotherapy. This may result in depletion of stem cells,organ failure and late effects that might contribute tomalignant transformation (3).Patients undergoing radiation are given hae

10、matopoieticcell therapy from three major sources: (i) autologous allogeneicBM, (ii) cytokine mobilized autologous peripheralblood, and recently (iii) cord blood. These sources of cellshave several limitations such as improper HLA-matcheddonor cells, requirement of immunosuppressant treatment,recurre

11、nce of tumours and requirement of high dosage ofcells for transplantation. The above limitations are clinicallymanifested as early effects (for example, mucositis,graft-versus-host disease, haemorrhagic cystitis, lunginjury and veno-occlusive disease) and late effects (forexample, musculoskeletal ef

12、fects, ocular effects, endocrineeffects and neurocognitive and neuropsychologicaleffects), thus demanding faster recovery of the haematopoieticsystem.Correspondence: A. Mukhopadhyay, Stem Cell Biology Laboratory,National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi-110067, India. Tel.: +9

13、1-11-26703781; Fax: +91-11-26702125; E-mail:ashoknii.res.in1Present address: Department of Neuroscience, School of MedicalSciences, University Sains Malaysia, Malaysia.10 _ 2010 Blackwell Publishing Ltd.Cell Prolif., 2011, 44, 1018 doi: 10.1111/j.1365-2184.2010.00713.xEarly recovery of the haematopo

14、ietic system fromlethal irradiation is strictly limited due to G0-silence orquiescence nature of HSCs. Hence, an alternative strategyis expected to be beneficial by engineering dormant haematopoieticcells for initiation of early signalling events.Although many HSC-specific transcription factors, gro

15、wthfactor receptors and corresponding ligands are known toactivate haematopoietic cells, recruitment of a master signallingswitch would ensure stage-specific regeneration ofcells (46).Previous studies have shown that viral vector-basedgene delivery in HSCs has many drawerbacks (79).Primitive HSCs (L

16、in)Sca-1+c-kit+; LSK), which areonly 0.5% of total BM cells, have densely packedDNA inside intact nuclear membranes. This rendersthem difficult to transfect by a viral-mediated genetransfer method. The second obstruction is seen aspost-maintenance of these modified populations in vivofor short-term

17、as well as long-term gene expression, asa result of competition between transduced and nontransducedcells. This has been shown in the case ofpatients with chronic granulomatous conditions infusedwith gp91phox-transduced purified CD34+ peripheralblood cells (7). The third and most severe problemassoc

18、iated with viral gene therapy is development ofoncogenesis arising from insertional mutagenesis. Manygene therapy studies have shown development ofleukaemia in patients who had undergone treatment forsevere combined immunodeficiency (8,9).Considering the above shortfalls of virus-mediatedgene transf

19、er, we propose a protocol of electroporationmediated,transient over-expression, of a master signaltransducer c-kitwt and its hyper-activating variant Kitmu(asp814tyr) to reconstitute the haematopoietic system.The electroporation method is known to induce lesscell death as cells do not experience che

20、mical toxicityas in the case of chemical-mediated transfection (1012). Earlier studies have confirmed c-kitwt as the keyreceptor for growth and survival of HSCs (13,14).Under normal circumstances, c-kit is activated by itsligand stem cell factor (SCF). In humans, constitutiveactivation of c-kit in n

21、eoplastic mastocytosis has beenshown to bypass SCF interference in downstream signallingdue to clustering of gain-of-function mutationAsp816 to Val in the Kit activation domain (15).Recently, we have predicted that certain activation loopresidues in c-kit are crucial for interaction of kinaseswith S

22、hp-1 for destabilization. Point mutation instretches of Lys818 to Ser 821 and Thr847 to Glu849in the Kit catalytic domain may lead to generation of ahyperactive variant of c-kit (16). In the mouse, Asp814to Tyr mutation of the Kit activation loop results inover-expression of kinases as by degradatio

23、n of tyrosinephosphatase (Shp-1) (17). Hyperactive Kit kinasehas been reported to be associated with constitutiveactivation of phosphoinositide-3 kinase (PI3 kinase),signal transducer and activator of transcription 3(STAT3), and nuclear factor kappa B (NF-jB) signallingpathways 1618. Cells affected

24、were also independentof SCF for growth (18). These results suggestthat recruitment of a hyper-activating mutant like Kitmu(asp814tyr) would influence downstream signalling inlethally irradiated hosts to facilitate haematopoiesis.Materials and methodsAnimalsSix- to eight-week-old C57BL 6J Ptprcb (Ly5

25、.2) andBL6.SJL Ptprcc (Ly5.1) mice were used in this study.Mice were obtained from the Jackson Laboratory (BarHarbor, Maine, USA, http:/www.jax.org) and maintainedin our institutes experimental animal facility. During theexperiments, mice were kept in an isolator and fed autoclavedacidified water an

26、d irradiated food ad libitum. Allexperiments using mice were conducted according to theprocedures approved by the Institutional Animal EthicsCommittee.Isolation of BM cellsBMCs were isolated by flushing two tibia and two femursfrom 6- to 8-week-old Ly5.1 mice, and erythrocytes werelysed by treatment

27、 with Geys solution (19). Cell suspensionwas incubated in a tissue culture plate for 12 h toremove adhered stromal cells. Prior to electroporation,non-adherent viable cells were counted using the trypanblue dye exclusion method.Electroporation of BM cellsBMCs were cultured for 12 h at 37 _C in a CO2

28、 incubator.Electroporation of cells (10 106 per cuvette) was performedusing a Gene Pulser II Biorad electroporator (Hercules,CA, USA) at 1.7 kVcm current and 400 ls pulselength. A custom-made minipulse chamber (with capacityof high cell density) having rectangular stainless steelelectrodes (4 mm) wa

29、s used for electroporation. Polyethyleneglycol-purified pcDNA3.1-Kit constructs werediluted in electroporation buffer 10 mM HEPES, IMDMsupplemented with 5% FCS and 5% FBS at a concentrationof 50100 lg DNA ml. To avoid post-pulse apoptosis,electroporated cells were washed and incubated at37 _C in the

30、 5% CO2 incubator for 15 min. Incubation ofpost-pulsed cells is believed to facilitate resealing of pores(10,11)._ 2010 Blackwell Publishing Ltd, Cell Proliferation, 44, 1018.Haematopoietic recovery by hyper-activating c-kit 11Culture of Ly5.1 BMCsElectroporated cells were cultured in Iscoves modifi

31、edDulbeccos medium (IMDM), supplemented with 10%FCS, 50 ng ml murine SCF and 10 ng ml interleukin-3(IL-3) (PeproTech Asia, Rehovot, Israel, http:/www.). Cells were cultured for different times at37 _C in a 5% CO2 incubator and viable cell number wasdetermined.Flow cytometryCells were labelled with antibodies and analysed accordingto methods described in our previous study (20).Antibodies used here were anti-: Sca-1 FITC, c-kit phycoerythrin-Cy5 (BD Pharmingen, San Jose, CA,USA), CD45.1 PE-Cy5 and CD16 32 (eBiosciences, SanDiego, CA, USA). Lineage a