CN1958092A - Method for promoting proliferation of hematopoietic cell - Google Patents
Method for promoting proliferation of hematopoietic cell Download PDFInfo
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- CN1958092A CN1958092A CN 200510116828 CN200510116828A CN1958092A CN 1958092 A CN1958092 A CN 1958092A CN 200510116828 CN200510116828 CN 200510116828 CN 200510116828 A CN200510116828 A CN 200510116828A CN 1958092 A CN1958092 A CN 1958092A
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Abstract
A method for promoting the reproduction of hematopoietic cell includes such steps as irradiating the bone marrow cells or hematopoietic stem cells of donor by low-dosage ray, and conventionally transplating the irradiated bone marrow cells into receptor.
Description
Technical field
The present invention relates to the method that hemopoietic function of bone marrow is rebuild, particularly relate to the method for utilizing the low dose radiation stimulating effect to promote bone marrow hematopoietic cell proliferation in the receptor.This method comprises in advance carries out external low dosage roentgenization to donor bone marrow cell or hematopoietic stem cell, in the donor bone marrow cell of then extracorporeal irradiation the being crossed input receptor.
Background technology
Since the eighties in 20th century, the hormesis of relevant low dose radiation research in the radiation medicine research field, having become one of focus.People stimulate cellular proliferation with regard to low dose radiation, enhancing human body immunity function etc. has carried out studying extensively and profoundly (Luckey TD.Nurture with ionizing radiation:a provocativehypothesis.Nutrition and cancer, 1999,34:1-11; Soviet Union sets a prairie fire, Du Zeji, and Hailin, field etc., low dose exposure is to lymphocytic effect, radiation research and radiation process journal, 2001,19:169-176; Liu Shuzheng, the some problems of low dose radiation hormesis genesis mechanism are inquired into, Chinese radiological medicine and protection magazine, 2003,23:393-398).The inventor's former research also confirms, be subjected to the peripheral blood of low dose exposure can produce tangible stimulating effect to the human blood cell who is subjected to high-dose irradiation, promote fast breeding (the tension force unit of hematopoietic cell, Jiang Jiagui, Lin Ping etc., the stimulating effect research of the stripped blood of low dose exposure, radiation research and radiation process journal, 2004,22:315-31; Jiang Jiagui etc., O
2 -Effect in cell proliferation stimulating effect due to the low-level radiation.Radiation research and radiation process journal, 2001,19:219-2237; The free radical mechanism of cell proliferation stimulating effect due to the Bai Haitao, Yi Jian, Jiang Jiagui, low-level radiation, Chinese radiological medicine and protection magazine, 2002,22:419-420).
Before the present invention, though existing many low dose radiations that studies show that have the effect of stimulating proliferation to hemopoietic system, up to now, still these donorcellses of nobody's proof are to the influence of receptor medulla hematopoietic system.
The inventor attempts utilizing low dose radiation to promote behind the bone marrow cell transplantation influence to receptor bone marrow hematopoietic reconstitution on existing basic research basis.The result fully proves, with low dose radiation stimulated in vitro donor bone marrow cell and after importing receptor, can promote the bone marrow hematogenesis of receptor to rebuild significantly.
Goal of the invention
The invention provides a kind of method that promotes myeloid element propagation, be characterised in that in advance transplanted donor bone marrow cell or hematopoietic stem cell are carried out external low dosage roentgenization, then the donor bone marrow cell of illuminated mistake is transplanted in the receptor of accepting bone marrow transplantation.
According to a preferred embodiment of the invention, wherein said low dosage ray is that dosage is the X ray of 6-8cGy.
Summary of the invention
The present invention relates to the method for myeloid element propagation, particularly relate to the method for utilizing the low dose radiation stimulating effect to promote receptor hematopoietic reconstitution after the bone marrow transplantation.
Briefly, method of the present invention comprises, before mice is carried out bone marrow transplantation, gives low dose exposure to the donor bone marrow suspension; Then, according to a conventional method with in the irradiated medullary cell input receptor; Peripheral blood cells and the BMNC quantity of input back monitoring receptor, with observe low dose radiation to bone marrow transplantation after the influence of animal hematopoietic reconstitution.The result confirms that low dose radiation can promote the bone marrow hematogenesis of receptor to rebuild really effectively.
Confirm further on the former basis of discovering of the inventor that after the medullary cell of low dose exposure carried out bone marrow transplantation, receptor BMNC number and peripheral blood cells counting generally were higher than corresponding matched group.Particularly, behind the low dose exposure group cell input receptor the 5th and 10 day, BMNC of receptor (BMMNC) and leukocyte (WBC) were apparently higher than matched group (P＜0.05).These experimental result promptings, low dose radiation may promote the propagation and the release of the medullary cell of transplant recipient Mus.
Since low dose radiation is to peripheral blood cells and the myeloid element effect of stimulating proliferation, so whether can before bone marrow transplantation, at first carry out external low dose exposure to donor bone marrow cell, to be subjected to the medullary cell of low dose irradiation to be transplanted to receptor more then? in other words, can low dose radiation promote the early stage hematopoietic reconstitution after mouse bone marrow cells is transplanted?
In order to confirm the influence of low dose exposure to the receptor hemopoietic function of bone marrow, the inventor uses living animal, has carried out following experimentation.
(1) at first, determine best radiation dose.With regard to human exposure, the radiation dose of low-level radiation generally be meant dosage 0.2Gy with interior pass at the low can line density (LET) radiation or dosage at 0.05Gy with interior high LET radiation, close rate is in 0.05mGy/ minute simultaneously.But in the practical study, usually exposure dose is met above-mentioned condition, close rate is higher than 0.05mGy/ minute person and is referred to as low dose radiation (LDR).Therefore, our research fixes on best radiation dose in the 10cGy.Radiation source adopt 6MV-X ray that the medical electronic linear accelerator often used clinically produces and
60The gamma-rays of Co emission.Adopt
3H-TdR mixes the multiplication capacity (seeing embodiment 1 for details) that method is weighed medullary cell.
(2) then, will import in the receptor through the donor bone marrow cell of low dose radiation.Be reliability that guarantees experiment and the rejection of avoiding cell input back receptor, we adopt homogenic BALB/C mice commonly used in the radiation medicine research as medullary cell donor and receptor model.In order to judge and the medullary cell stimulation of check analysis low dose radiation more objective and accurately to transplanting, and the medullary cell extracorporeal irradiation is to the influence of receptor hemopoietic function of bone marrow, and laboratory animal is divided into four groups: normal group, normally transplant matched group, 6cGy irradiation transplantation group and 8cGy irradiation transplantation group (seeing embodiment 2 for details).
At last, the peripheral blood cells of animal counting is judged the influence that low dose radiation is rebuild the receptor bone marrow hematogenesis by experiment.The peripheral blood cells quantity of receptor is reliable, the most direct, the most classical index of reflection bone marrow transplantation effect.Therefore, we are carrying out effect observation, the number change of emphasis monitoring peripheral hemocyte, also dynamic observe simultaneously the mononuclearcell number change of mouse femur, and the spleen tuberosity quantity the 10th day time the, and the general situation of medullary cell receptor (active degree and food-intake) (seeing embodiment 3 for details).
The inventor has proved the facilitation of the stimulating effect of low dose radiation to receptor bone marrow hematopoietic reconstitution first.The inventive method is characterised in that, at first donor bone marrow cell or hematopoietic stem cell carried out the low dose irradiation of 6～8cGy, and then in the medullary cell input receptor with illuminated mistake.Because we carry out external low dose irradiation with the medullary cell from donor, therefore can not cause any harmful effect to donor.
The inventor confirms that through isolated experiment (6～10cGy) can impel the bone marrow cells in mice proliferation activity to improve to low dose radiation, promote cell growth and breeding.In general, employed radiation dose scope is 6～10cGy, preferably 6～8cGy.If the radiation dose that uses is too high, ionizing radiation produces certain cellulotoxic effect on the contrary, causes multiplication effect to weaken.
Animal experiment study shows, will (behind the medullary cell input receptor of 6～8cGy) irradiations, the hemopoietic function of bone marrow of receptor be significantly improved through low dosage.The general physiological situation that is in particular in BMNC number, peripheral blood cells counting, spleen tuberosity quantity and the receptor mice of the every femur of receptor mice is all significantly improved or is improved.
Therefore, our conclusion is that (6～8cGy) medullary cell or hematopoietic stem cell can promote the early stage hematopoietic reconstitution of receptor effectively through low dose radiation.
The specific embodiment
Embodiment 1: the radiation dose of determining to produce the optimal stimulus effect
The bone marrow cell suspension preparation; With average weight is after the BALB/C male mice dislocation of 18-22g is put to death, to place 75% alcohol-pickled 3-5 minute.Then, clip bilateral femur under aseptic condition, and draw no phenol red RPMI-1640 culture fluid with the syringe that has No. 7 syringe needles and wash conventional isolated mononuclear cell after the medullary cavity.Behind peripheral blood cells calculating instrument (Beckmen Co.) counting BMNC number, cell density is adjusted into 1 * 10
6/ mL.
The 6MV x-ray bombardment donor bone marrow cell that adopts linear accelerator (Philips Co.) to produce.Source-skin distance (SSD) used during irradiation is 100cm, and field size is 10cm * 10cm, and the low dose exposure close rate is 3cGy/ minute.
Get 21 culture bottles, add RPMI-1640 culture fluid 2mL in every bottle, cell suspension 0.1mL
3The thymidylic acid of H labelling (
3H-TdR) 20 μ L (2.22 * 10
5Bq/mL).Above-mentioned culture bottle shakes up rearmounted 37 ℃ and cultivated 1,2,3,4,5,6 and 7 hour, and puts 4 ℃ in 3 parallel sample bottles of each time point taking-up and deposit.All cultivate finish after, by the filter membrane method sample preparation, survey the CPM value (numeration of per minute radioactive decay) of each bottle with liquid glimmer instrument, with definite best incubation time point (referring to table 1).
Other gets 18 culture bottles, adds no phenol red RPMI-1640 culture fluid 2mL and cell suspension 0.1mL in every bottle.They are divided into six groups: normal control group, 2cGy, 4cGy, 6cGy, 8cGy and 10cGy irradiation group (per 3 culture bottles are a dose point).After culture bottle was accepted x-ray bombardment, every bottle added
3H-TdR20 μ L.Cultivate after 3 hours, use the filter membrane method sample preparation and survey the CPM value of each bottle with liquid glimmer instrument.Each experiment in above-mentioned two stages experiment all repeats 5 times (table 2).
The result is with (the expression of x ± s), and adopt SPSS 10.0 softwares to carry out variance analysis.Shown in following tabulation 1 of result and the table 2.
Table 1. bone marrow cells in mice is at different incubation times
3The H-TdR value of mixing
| Group | Incubation time (hour) | ? 3H-TdR value of mixing (CPM) |
| I II III IV V VI VII | 1 2 3 4 5 6 7 | 3207±436.2 3783±612.2 4815±421.3 * 4209±369.6 ▲ 3986±489.6 # 3923±398.5 ## 3298±326.3 |
*Compare P＜0.001 with the sample of cultivating 1 hour;
▲Compare P＜0.05 with the sample of cultivating 1 hour;
#Compare P＜0.05 with the sample of cultivating 1 hour;
##Compare P＜0.05 with the sample of cultivating 1 hour.
By table 1 as seen, normal mouse medullary cell isolated culture 3 hours, the CPM value is the highest.Therefore, we will continue after cell culture time in every experiment all be defined as 3 hours.
Table 2. bone marrow cells in mice is behind the different low dose exposures
3The H-TdR value of mixing
| Group | Exposure dose (cGy) | ? 3H-TdR value of mixing (CPM) |
| I II III IV V VI | 0 2 4 6 8 10 | 4815±421.3 5136±312.2 5389±521.3 6809±569.9 * 6081±789.5 # 5626±621.6 ## |
*Compare P＜0.01 with the normal control group;
#Compare P＜0.05 with the normal control group;
##Compare P＜0.05. with the normal control group
Data declaration shown in the table 2, although 6cGy, 8cGy and 10cGy irradiation all can cause bone marrow cells in mice propagation to be accelerated, the multiplication effect when 6cGy and 8cGy is the most obvious.Compare with the normal control group, the CPM value of 6cGy and 8cGy group obviously increases (P＜0.01).When radiation dose was too high, ionizing radiation may produce certain cellulotoxic effect, caused cell proliferation to weaken.Therefore, we select 6cGy and 8cGy as optimal stimulus dosage.
Embodiment 2: irradiated donor bone marrow cell is to the influence of receptor hematopoietic reconstitution
The preparation of bone marrow cell suspension: the BALB/C male mice (being provided by University Of Suzhou zoopery center) that is averaged body weight and is 18-22g is as donor animal, began to drink in 3 days in pre-irradiation and contain gentamycin (320mg/L)+erythromycin (250mg/L) water for injection, up to transplanting 2 weeks of back.The receptor Mus is female cleaning level BALB/C mice.
Animal is put to death in dislocation, after 75% alcohol-pickled 3-5 minute, aseptic clip bilateral femur, draw no phenol red RPMI-1640 culture fluid flushing medullary cavity and isolate individual cells with the syringe that has No. 7 syringe needles, use blood-counter system (Beckmen Co.) to measure the BMNC number of every femur by the leukocytic method of counting then.
The 6MV X ray that adopts linear accelerator (Philips Co.) to produce carries out low dose exposure, and the source-skin distance when wherein shining (SSD) is 100cm, irradiation field 10cm * 10cm, and the close rate of low dose exposure is 3cGy/ minute.High-dose irradiation adopts
60Co is as radiation source.
Laboratory animal is divided into 4 groups at random: 5 of normal group, do not do any processing; 20 of the matched groups (hereinafter to be referred as matched group) that the employing normal marrow cell is transplanted; 20 of the processed group (hereinafter to be referred as 6cGy irradiation group) that adopt the postradiation medullary cell of 6cGy to transplant; 20 of the processed group (hereinafter to be referred as 8cGy irradiation group) that adopt the postradiation medullary cell of 8cGy to transplant.
Then according to steps of processing: 1. irradiation: adopt cover to use according to mode
60It is 7.5Gy that Co carries out whole body uniform irradiation dosage to the receptor mice, and close rate is 1Gy/ minute.2. the foundation of model: get 10 of healthy normal male BALB/c mouse at every turn, after the cervical vertebra dislocation is put to death, get the bilateral femur immediately and prepare bone marrow nucleated cell suspension and counting, cell concentration is adjusted to 5 * 10
6/ mL.Divide 3 groups with the cell suspension for preparing, promptly accept two the irradiation groups (6cGy irradiation group and 8cGy irradiation group) and the false irradiation group (matched group) of 6cGy and 8cGy (close rate is 3cGy/ minute).With above-mentioned three groups of cell suspension respectively at (the input cell number is 2 * 10 in the mice body after 1. the input of tail vein is handled by above-mentioned steps in 4 hours
6/ only, amount of liquid 0.4mL/ is only).
Then, observe and write down peripheral blood cells counting, (every femur) BMNC (BMMNC) counting, splenocyte colony forming unit (CFU-S) counting of the receptor of input medullary cell, and general behavior situation (active degree and food consumption quantity), accept the influence of the receptor bone marrow hematogenesis being rebuild behind the low dose exposure so as to judging donor bone marrow cell.
Behind the cell input receptor the 5th, 10,15,30 days, 5 of picked at random and collect carotid artery blood from 6cGy irradiation group, 8cGy irradiation group and control animals respectively.After oxalic acid dipotassium anticoagulant processing, with blood-counter system (Beckmen Co.) counting peripheral blood leucocyte, erythrocyte and platelet count.
Then, putting to death animal and immersing volume fraction is to place 5 minutes in 75% ethanol, asepticly gets complete femur and washes out mononuclearcell with RPMI 1640 liquid from medullary cavity, counts by the numeration of leukocyte method.
Simultaneously, put to death animal on the 10th day to get spleen, in 5%Bouin solution fix 24 hours and with 75% ethanol decolorization after, under naked eyes, make spleen convex surface tuberosity and count.
In addition, before putting to death animal, observe and write down active degree and the food-intake of experiment made on the living animal.
The result with (expression of x ± s) and adopt SPSS 10.0 softwares carry out variance analysis (referring under tabulate 3).
Table 3: peripheral blood WBC, RBC, Plt and BMMNC counting
| Group | Observing time (my god) | The Mus number | BMMNC ? (×10 6/ bone) | WBC ? (×10 9/L) | RBC ? (×10 12/L) | Plt ? (×10 9/L) |
| Normal group control group 6cGy irradiation group 8cGy irradiation group control group 6cGy irradiation group 8cGy irradiation group control group 6cGy irradiation group 8cGy irradiation group control group 6cGy irradiation group 8cGy irradiation group | - 5 5 5 10 10 10 15 15 15 30 30 30 | 10 10 10 10 10 10 10 10 10 10 10 10 10 | 4.41±0.71 0.28±0.05 0.45±0.07 * 0.42±0.06 # 0.49±0.08 0.71±0.08 * 0.65±0.07 * 0.77±0.12 0.99±0.13 0.97±0.13 3.68±1.00 4.69±1.06 4.15±0.94 | 6.6±1.49 0.34±0.08 0.57±0.09 * 0.54±0.08 # 0.60±0.08 0.82±0.09 * 0.76±0.12 1.22±0.18 1.59±0.14 * 1.59±0.23 # 1.16±0.16 1.56±0.12 * 1.36±0.19 | 8.2±0.91 7.1±1.35 6.9±1.26 6.5±1.22 6.41±1.31 6.86±1.19 6.95±1.13 6.68±1.03 7.67±0.90 7.84±1.15 7.5±0.98 7.73±1.01 7.41±0.97 | 805.4±162.7 48.2±22.1 77.6±28.1 66.6±24.9 72.2±24.8 125±36.6 117.8±34.5 117.8±41.9 202.1±40.2 197.1±47.9 313.3±34.2 466.2±88.0 # 400.6±79.2 |
Annotate: compare with corresponding matched group,
*P＜0.01;
#P＜0.05;
Result shown in the table 3 shows, uses behind two groups of medullary cells input receptors of low dose exposure, and every femur bone marrow mononuclearcell number of two groups of receptors and peripheral blood cells counting are apparently higher than corresponding matched group (being conventional transplantation group).Statistical test result shows, this two treated animal the 5th and 10 day behind the input donor bone marrow cell, and BMMNC and WBC digital display work are higher than matched group (P＜0.01 or 0.05).
Observe from the RBC angle, the RBC counting of two treated animals of low dose exposure cell does not have significant difference with matched group.As for Plt, though the irradiation two groups all apparently higher than matched group since each the group standard deviation too big, statistics relatively shows only significant difference (P＜0.05) in the time of the 30th day.
The CFU-S counting of control group mice is 8.2 ± 2.82; 6cGy irradiation group mice CFU-S counting is 12.1 ± 2.73; 8cGy irradiation group is 11.5 ± 2.79.The CFU-S counting of two groups of mices of low dose exposure is significantly more than matched group (P＜0.05).In addition, it is obviously active than matched group that experimental session is observed the mice of 6cGy and 8cGy irradiation group, and preceding two groups food consumption quantity is significantly greater than the latter.
Claims (2)
1, promote the method for hematopoietic cell proliferation in the receptor, be characterised in that in advance donor bone marrow cell or hematopoietic stem cell are carried out the low dosage roentgenization, and then in the donor bone marrow cell input receptor with illuminated mistake.
2, according to the process of claim 1 wherein that said low dosage ray is that dosage is the X ray of 6-8cGy.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010124585A1 (en) * | 2009-04-30 | 2010-11-04 | Adistem Ltd | Methods and apparatuses for isolating and preparing stem cells |
| CN102264890A (en) * | 2009-01-27 | 2011-11-30 | 株式会社Jms | Method for controlling propagation of umbilical cord blood hematopoietic stem cells and use thereof |
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2005
- 2005-10-31 CN CN 200510116828 patent/CN1958092A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102264890A (en) * | 2009-01-27 | 2011-11-30 | 株式会社Jms | Method for controlling propagation of umbilical cord blood hematopoietic stem cells and use thereof |
| CN102264890B (en) * | 2009-01-27 | 2013-05-01 | 株式会社Jms | Method for controlling propagation of umbilical cord blood hematopoietic stem cells and use thereof |
| WO2010124585A1 (en) * | 2009-04-30 | 2010-11-04 | Adistem Ltd | Methods and apparatuses for isolating and preparing stem cells |
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