CN108753719B - A method of the LAK LAK of derived from cord blood is improved to tumor-killing power - Google Patents
A method of the LAK LAK of derived from cord blood is improved to tumor-killing power Download PDFInfo
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- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
- C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
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Abstract
The invention discloses a kind of LAK LAK for improving derived from cord blood to the method for tumor-killing power, and cord blood mononuclear cells is modulated with 1640 culture medium of RPMI containing 10% fetal calf serum to 2 × 106/ mL, is inoculated in the culture vessel of different capabilities according to total number of cells, while α-ring costunolide of final concentration of 20-30 μ g/mL or the rIL-2 of β-ring costunolide and 500IU/mL is added, and is placed in 37 DEG C, 5%CO2Under the conditions of cultivate, measure 1640 culture medium of 10%FCS-RPMI of replacement rIL-2 containing comparable sodium every 2~3d half.After the method for the present invention halves rIL-2 on the basis of existing method, there is no reduce LAK cell to the killing rate of liver cancer cells, on the contrary, the killing rate to liver cancer cells significantly improves.
Description
Technical field
The invention belongs to biological field, it is immune to be related to tumour cell, and in particular to a kind of lymph for improving derived from cord blood because
Method of the son activation killing cell LAK to tumor-killing power.
Background technique
Immunization therapy is a kind of emerging oncotherapy mode, has been listed in the 4th kind after operation, radiotherapy, chemotherapy
Therapeutic modality gradually plays a significant role in the comprehensive treatment of tumors.2010, U.S. FDA approval was first thin in the world
Born of the same parents' immunotherapeutic product Provenge listing, sufficiently shows value of the cellular immunotherapy in oncotherapy.
LAK (LAK) is in the 1980s by research and development such as Rosenberg, its essence is
IL-2 activation has the NK and T cell for killing tumor activity.In November, 1984, Rosenberg study group ratified through U.S. FDA, for the first time
LAK cell is used for clinical treatment.The therapy is to metastatic renal cell cancer, melanoma, colon cancer and non-Hodgkin lymphoma
The curative effect of patient is more significant.But LAK lethality is not strong, and clinical application needs a large amount of infusions.And its amplification ability is limited, needs
Large dosage application IL-2 while infused cells, so as to cause apparent toxic side effect, most common and most serious poison is secondary to be made
With being capillary leak syndrome, be mainly shown as anasarca and multiple organ dysfunction imbalance, can cause pleural effusions and ascites,
Pulmonary interstitial edema and congestive heart failure.
Therefore, the extensive use of LAK cell is firstly the need of solving two problems: first, how to improve the killing of LAK cell
Power；Second, the amplification ability of LAK cell how is improved under the premise of little dose-dependant IL-2.
Summary of the invention
The present invention is directed to overcome the shortage of prior art, a kind of LAK for improving derived from cord blood is provided
Method of the LAK to tumor-killing power.
Technical solution of the present invention is as follows:
A kind of LAK LAK improving derived from cord blood to the extracorporeal culturing method of tumor-killing power,
RIL-2 is added in culture medium, is also added with α-ring costunolide or β-ring costunolide.
Preferably, the extracorporeal culturing method specific steps are as follows:
Separating umbilical blood mononuclearcell, by RPMI 1640 culture medium of the cord blood mononuclear cells containing 10% fetal calf serum
(10%FCS-RPMI 1640) is modulated to 2 × 106/ mL, is inoculated in the culture vessel of different capabilities according to total number of cells, together
When α-ring costunolide of final concentration of 20-30 μ g/mL or the rIL-2 of β-ring costunolide and 500IU/mL is added, set
In 37 DEG C, 5%CO2Under the conditions of cultivate, trained every the 10%FCS-RPMI 1640 that 2~3d half measures replacement rIL-2 containing comparable sodium
Support base.
Preferably, thin using the ficoll cardiografin layering single core of liquid density-gradient centrifugation method separating umbilical blood from bleeding of the umbilicus
Born of the same parents.
Preferably, cord blood collection method are as follows: full-term normal delivery healthy newborn bleeding of the umbilicus is acquired using closed collecting method, and
Its parent of antenatal exaination is without genetic disease family history, using the anticoagulant closed blood bag of citrate-phosphate-glucose in tire
Disk acquires before giving birth to from the umbilical cord broken ends of fractured bone far from newborn side；4 DEG C of refrigerators save after acquisition, interior for 24 hours to be separated and frozen processing
It is spare.
Preferably, the tumour is liver cancer.
A kind of culture medium of the LAK LAK of derived from cord blood, in the RPMI for containing 10% fetal calf serum
α-ring the costunolide or β-ring costunolide and 500IU/mL of final concentration 20-30 μ g/mL are added in 1640 culture mediums
rIL-2。
Application of the above-mentioned culture medium in terms of the LAK LAK culture of derived from cord blood.
A method of measurement LAK cell is to liver cancer cells lethality, specific steps are as follows:
It takes the LAK cell of Fiber differentiation 10d as effector cell, using HepG2 liver cancer cells as target cell, imitates target by 20:1
Than 96 orifice plates are added in effector cell and target cell respectively, independent target cell and individual effect groups of cells are separately set, every group sets 3 again
37 DEG C, 5%CO are placed in hole2After cultivating for 24 hours in incubator, every hole adds 10 μ L of MTT (5mg/mL), continues to cultivate 4h, be centrifuged on abandoning
Clearly, after every hole adds 150 μ L of dimethyl sulfoxide, oscillation to dissolve 10min, light absorption value A is surveyed with microplate reader 570nm, is counted as follows
Each group LAK cell is calculated to the killing activity of liver cancer cells；
Killing rate (%)=[1- (experimental group A value-individual effect cell A value)/independent target cell A value] × 100%.
The utility model has the advantages that
After the method for the present invention halves rIL-2 on the basis of existing method, there is no reduce LAK cell to liver cancer cells
Killing rate, on the contrary, killing of the LAK cell under α-ring costunolide or the induction of β-ring costunolide, to liver cancer cells
Rate significantly improves, and difference has statistical significance (P < 0.05)；In addition, α-ring costunolide or β-ring costunolide pair
There are apparent dose dependents for the inducing action of LAK cell.
Detailed description of the invention
Fig. 1 is killing rate (%) of each group LAK cell to liver cancer cells.
Specific embodiment
It is specific with reference to the accompanying drawings and examples to introduce essentiality content of the present invention, but guarantor of the invention is not limited with this
Protect range.
Embodiment 1:
One, experimental material
α-ring costunolide (No. CAS: 2221-81-0), β-ring costunolide (No. CAS: 2221-82-1), γ-ring
The purity of costunolide (No. CAS: 24164-19-0) is not less than 98%.
1640 culture medium of RPMI, 10% fetal calf serum are purchased from Gibco company.
RIL-2 is purchased from Beijing Fourth Ring biopharmaceutical company.
Two, experimental method
1, cord blood collection and mononuclearcell separation
Full-term normal delivery healthy newborn bleeding of the umbilicus 100 is acquired using closed collecting method, and its parent of antenatal exaination is equal
Without genetic disease family history, using the anticoagulant closed blood bag of citrate-phosphate-glucose (CPDA) before placenta is given birth to from
The umbilical cord broken ends of fractured bone is acquired far from newborn side.4 DEG C of refrigerators save after acquisition, interior for 24 hours to be separated and frozen processing.
With reference to art technology handbook (Shen Guanxin etc., Immunology Today experimental technique, Hubei science tech publishing house,
Page 1998,172-175), liquid density-gradient centrifugation method separating umbilical blood mononuclearcell is layered using ficoll cardiografin.
2, the Fiber differentiation of LAK cell
(1) existing method
By RPMI 1640 culture medium (10%FCS-RPMI of the above-mentioned cord blood mononuclear cells containing 10% fetal calf serum
1640) it modulates to 2 × 106/ mL, is inoculated in the culture vessel of different capabilities according to total number of cells, while being added final concentration of
The phytolectin of 60 μ g/mL and the rIL-2 of 1000IU/mL, are placed in 37 DEG C, 5%CO2Under the conditions of cultivate, measured every 2~3d half
Replace 1640 culture medium of 10%FCS-RPMI of the rIL-2 containing comparable sodium.
(2) the method for the present invention
By RPMI 1640 culture medium (10%FCS-RPMI of the above-mentioned cord blood mononuclear cells containing 10% fetal calf serum
1640) it modulates to 2 × 106/ mL, is inoculated in the culture vessel of different capabilities according to total number of cells, while being added final concentration of
The rIL-2 of the α of 25 μ g/mL-ring costunolide, β-ring costunolide or γ-ring costunolide and 500IU/mL, is placed in
37 DEG C, 5%CO2Under the conditions of cultivate, cultivated every the 10%FCS-RPMI 1640 that 2~3d half measures replacement rIL-2 containing comparable sodium
Base.
(3) control methods (only adding rIL-2)
By RPMI 1640 culture medium (10%FCS-RPMI of the above-mentioned cord blood mononuclear cells containing 10% fetal calf serum
1640) it modulates to 2 × 106/ mL, is inoculated in the culture vessel of different capabilities according to total number of cells, while being added final concentration of
The rIL-2 of 500IU/mL is placed in 37 DEG C, 5%CO2Under the conditions of cultivate, measure replacement rIL-2's containing comparable sodium every 2~3d half
1640 culture medium of 10%FCS-RPMI.
3, tumor cell killing potential is detected
HepG2 cell lines freeze for our company, are cultivated after recovery using the RPMI 1640 containing 10% fetal calf serum
Base is placed in 37 DEG C, 5%CO2Secondary culture in incubator takes logarithmic phase to carry out subsequent experimental.
Take the LAK cell of each group Fiber differentiation 10d as effector cell, using HepG2 liver cancer cells as target cell, by 20:1
Target is imitated than 96 orifice plates are added in effector cell and target cell respectively, separately sets independent target cell and individual effect groups of cells, every group sets 3
A multiple holes place 37 DEG C, 5%CO2After cultivating for 24 hours in incubator, every hole adds 10 μ L of MTT (5mg/mL), continues to cultivate 4h, centrifugation is abandoned
Supernatant after every hole adds 150 μ L of dimethyl sulfoxide, oscillation to dissolve 10min, surveys light absorption value A with microplate reader 570nm, as follows
Each group LAK cell is calculated to the killing activity of liver cancer cells.
Killing rate (%)=[1- (experimental group A value-individual effect cell A value)/independent target cell A value] × 100%
4, statistical procedures
Data are indicated using mean value ± standard deviation, are examined with SPSS17.0 statistical software row t, and P < 0.05 has statistics
Meaning.
Three, experimental result
Each group LAK cell is to the killing rate of liver cancer cells as shown in table 1 and Fig. 1.Should the experimental results showed that, the method for the present invention
After halving rIL-2 on the basis of existing method, there is no reduce LAK cell to the killing rate of liver cancer cells, on the contrary, LAK is thin
Born of the same parents significantly improve (P < to the killing rate of liver cancer cells under α-ring costunolide or the induction of β-ring costunolide
0.05)。
Killing rate of the 1 each group LAK cell of table to liver cancer cells
Embodiment 2:
One, experimental material
α-ring costunolide, β-ring costunolide purity are not less than 98%.
1640 culture medium of RPMI, 10% fetal calf serum are purchased from Gibco company.
RIL-2 is purchased from Beijing Fourth Ring biopharmaceutical company.
Two, experimental method
1, cord blood collection and mononuclearcell separation
Full-term normal delivery healthy newborn bleeding of the umbilicus 100 is acquired using closed collecting method, and its parent of antenatal exaination is equal
Without genetic disease family history, using the anticoagulant closed blood bag of citrate-phosphate-glucose (CPDA) before placenta is given birth to from
The umbilical cord broken ends of fractured bone is acquired far from newborn side.4 DEG C of refrigerators save after acquisition, interior for 24 hours to be separated and frozen processing.
With reference to art technology handbook (Shen Guanxin etc., Immunology Today experimental technique, Hubei science tech publishing house,
Page 1998,172-175), liquid density-gradient centrifugation method separating umbilical blood mononuclearcell is layered using ficoll cardiografin.
2, the Fiber differentiation of LAK cell
By RPMI 1640 culture medium (10%FCS-RPMI of the above-mentioned cord blood mononuclear cells containing 10% fetal calf serum
1640) it modulates to 2 × 106/ mL, is inoculated in the culture vessel of different capabilities according to total number of cells, while being added final concentration of
The rIL-2 of the α of 20 μ g/mL-ring costunolide or β-ring costunolide and 500IU/mL, is placed in 37 DEG C, 5%CO2Condition
Lower culture measures 1640 culture medium of 10%FCS-RPMI of replacement rIL-2 containing comparable sodium every 2~3d half.
3, tumor cell killing potential is detected
HepG2 cell lines freeze for our company, are cultivated after recovery using the RPMI 1640 containing 10% fetal calf serum
Base is placed in 37 DEG C, 5%CO2Secondary culture in incubator takes logarithmic phase to carry out subsequent experimental.
Take the LAK cell of each group Fiber differentiation 10d as effector cell, using HepG2 liver cancer cells as target cell, by 20:1
Target is imitated than 96 orifice plates are added in effector cell and target cell respectively, separately sets independent target cell and individual effect groups of cells, every group sets 3
A multiple holes place 37 DEG C, 5%CO2After cultivating for 24 hours in incubator, every hole adds 10 μ L of MTT (5mg/mL), continues to cultivate 4h, centrifugation is abandoned
Supernatant after every hole adds 150 μ L of dimethyl sulfoxide, oscillation to dissolve 10min, surveys light absorption value A with microplate reader 570nm, as follows
Each group LAK cell is calculated to the killing activity of liver cancer cells.
Killing rate (%)=[1- (experimental group A value-individual effect cell A value)/independent target cell A value] × 100%
4, statistical procedures
Data are indicated using mean value ± standard deviation, are examined with SPSS17.0 statistical software row t, and P < 0.05 has statistics
Meaning.
Three, experimental result
The experimental results showed that when α-ring costunolide or the final concentration of 20 μ g/mL of β-ring costunolide, Fiber differentiation
The LAK cell of 10d is respectively (65.1 ± 6.3) %, (62.8 ± 6.5) % to the killing rate of HepG2 liver cancer cells.
Embodiment 3:
One, experimental material
α-ring costunolide, β-ring costunolide purity are not less than 98%.
1640 culture medium of RPMI, 10% fetal calf serum are purchased from Gibco company.
RIL-2 is purchased from Beijing Fourth Ring biopharmaceutical company.
Two, experimental method
1, cord blood collection and mononuclearcell separation
Full-term normal delivery healthy newborn bleeding of the umbilicus 100 is acquired using closed collecting method, and its parent of antenatal exaination is equal
Without genetic disease family history, using the anticoagulant closed blood bag of citrate-phosphate-glucose (CPDA) before placenta is given birth to from
The umbilical cord broken ends of fractured bone is acquired far from newborn side.4 DEG C of refrigerators save after acquisition, interior for 24 hours to be separated and frozen processing.
With reference to art technology handbook (Shen Guanxin etc., Immunology Today experimental technique, Hubei science tech publishing house,
Page 1998,172-175), liquid density-gradient centrifugation method separating umbilical blood mononuclearcell is layered using ficoll cardiografin.
2, the Fiber differentiation of LAK cell
By RPMI 1640 culture medium (10%FCS-RPMI of the above-mentioned cord blood mononuclear cells containing 10% fetal calf serum
1640) it modulates to 2 × 106/ mL, is inoculated in the culture vessel of different capabilities according to total number of cells, while being added final concentration of
The rIL-2 of the α of 30 μ g/mL-ring costunolide or β-ring costunolide and 500IU/mL, is placed in 37 DEG C, 5%CO2Condition
Lower culture measures 1640 culture medium of 10%FCS-RPMI of replacement rIL-2 containing comparable sodium every 2~3d half.
3, tumor cell killing potential is detected
HepG2 cell lines freeze for our company, are cultivated after recovery using the RPMI 1640 containing 10% fetal calf serum
Base is placed in 37 DEG C, 5%CO2Secondary culture in incubator takes logarithmic phase to carry out subsequent experimental.
Take the LAK cell of each group Fiber differentiation 10d as effector cell, using HepG2 liver cancer cells as target cell, by 20:1
Target is imitated than 96 orifice plates are added in effector cell and target cell respectively, separately sets independent target cell and individual effect groups of cells, every group sets 3
A multiple holes place 37 DEG C, 5%CO2After cultivating for 24 hours in incubator, every hole adds 10 μ L of MTT (5mg/mL), continues to cultivate 4h, centrifugation is abandoned
Supernatant after every hole adds 150 μ L of dimethyl sulfoxide, oscillation to dissolve 10min, surveys light absorption value A with microplate reader 570nm, as follows
Each group LAK cell is calculated to the killing activity of liver cancer cells.
Killing rate (%)=[1- (experimental group A value-individual effect cell A value)/independent target cell A value] × 100%
4, statistical procedures
Data are indicated using mean value ± standard deviation, are examined with SPSS17.0 statistical software row t, and P < 0.05 has statistics
Meaning.
Three, experimental result
The experimental results showed that when α-ring costunolide or the final concentration of 30 μ g/mL of β-ring costunolide, Fiber differentiation
The LAK cell of 10d is respectively (80.2 ± 6.7) %, (79.5 ± 6.9) % to the killing rate of HepG2 liver cancer cells.
Embodiment 4:
A kind of culture medium of the LAK LAK of derived from cord blood, in the RPMI for containing 10% fetal calf serum
α-ring the costunolide or β-ring costunolide and 500IU/mL of final concentration 20-30 μ g/mL are added in 1640 culture mediums
rIL-2。
To sum up, after the method for the present invention halves rIL-2 on the basis of existing method, there is no reduce LAK cell pair
The killing rate of liver cancer cells, on the contrary, LAK cell is under α-ring costunolide or the induction of β-ring costunolide, to liver cancer
The killing rate of cell significantly improves, and difference has statistical significance (P < 0.05)；In addition, α-ring costunolide or β-ring wood
There are apparent dose dependents for inducing action of the fragrant alkene lactone to LAK cell.
Above-described embodiment is intended to specifically introduce essentiality content of the present invention, but protection scope of the present invention is confined to this specifically
Embodiment.
Claims (2)
1. the culture medium of the LAK LAK of derived from cord blood a kind of, it is characterised in that: containing 10% tire ox blood
α-ring the costunolide and final concentration of 500IU/mL of final concentration of 20-30 μ g/mL are added in clear 1640 culture medium of RPMI
RIL-2, or β-ring radix aucklandiae of final concentration of 20-30 μ g/mL is added in 1640 culture medium of RPMI containing 10% fetal calf serum
The rIL-2 of alkene lactone and final concentration of 500IU/mL.
2. application of the culture medium described in claim 1 in terms of the LAK LAK culture of derived from cord blood.
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