CN1417583A - Detection method of renal bone damage caused by radiant mineral salt - Google Patents
Detection method of renal bone damage caused by radiant mineral salt Download PDFInfo
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- CN1417583A CN1417583A CN 02150739 CN02150739A CN1417583A CN 1417583 A CN1417583 A CN 1417583A CN 02150739 CN02150739 CN 02150739 CN 02150739 A CN02150739 A CN 02150739A CN 1417583 A CN1417583 A CN 1417583A
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Abstract
The present invention belongs to the field of radiobiology and bone biology, and is especially the detection method of renal bone damage caused by radiant mineral salt. Cs,y ray is used to radiate renal area to cause kidney damage and mineral salt content in bone and bone structure change are detected to establish radiant renal bone disease detecting method and research the pathological mechanism, preventing and treatment method of bone mineral salt damage induced by radiotherapy on the renal area. Through the research of substance of Chinese medicine "kidney controlling bone" theory and kidney invigorating mechanism, the present invention can provide new technological measures for the research of bone mineral salt metabolism mechanism and relevant diseases.
Description
Technical field
The invention belongs to radiobiology and bone biology field, be specifically related to a kind of detection radiation kidney bone mineral salt damage method
Background technology:
Kidney district partial radiation such as accident abdominal irradiation, radiation therapy etc., easily bring out renal dysfunction, renal dysfunction can cause bone mineral content damage to sclerotin, causes osteoporosis and osteomalacia in various degree, and its key link is the bad bone injury that causes of VD.The method of detection renal osteodystrophy sclerotin injury commonly used has kidney vascular ligation or kidney partly to excise back osteopathy reason norphometry inspection at present, and the X-ray plain film is checked and bone densitometry.Said method detects the step complexity, costs an arm and a leg, and the renal osteodystrophy bone is decreased pathological change and mechanism and drug therapy can not further investigate.
Summary of the invention
The objective of the invention is to set up radiation renal osteodystrophy detection method by detecting the change of bone mineral content amount and matter.This method can be studied kidney district radiation insult and bring out the pathomechanism of bone mineral content damage and prevent and treat method, and the method and the mechanism of theoretical essence of the research traditional Chinese medical science " kidney master bone " and kidney tonifying treatment bone mineral content disease.
The present invention utilizes
137Cs radiation gamma kidney district causes kidney injury, by detecting the change of bone mineral content amount and matter, sets up radiation renal osteodystrophy detection method.The present invention sets up radiation kidney bone mineral salt damage detecting method by following step.
1. kidney irradiation, get that side direction exposes its both sides kidney under the animal used as test SD rat anesthesia state, and the animal lateral position is fixed on the mould, use thickness 4.2cm, the stereotype of ray attenuation 98% blocks the tissue beyond the kidney, and tissue is shone beyond reaching accurate location and avoiding kidney.The irradiation metering is 10-50Gy.
2. injury of kidney detects:
Blood urea nitrogen, creatinine dynamic monitoring: per 2 week the tail vein get blood, automatic biochemistry analyzer is measured concentration.
Urine amount, urine protein, UCr clearance rate, the dynamic monitoring of urine β2Wei Qiudanbai: per 2 week is collected twenty-four-hour urine liquid, measure the urine amount, automatic biochemistry analyzer mensuration urine protein and concentration of urinary creatinine are (according to 24h urine amount, serum creatinine, the value of UCr is calculated CrCl), the concentration of measured by radioimmunoassay β2Wei Qiudanbai.
VD detects: 1,25 (OH) in the serum
2D
3Level can reflect kidney 1 α hydroxylase activity indirectly.Per vein collection end of the month serum 0.6ml, measured by radioimmunoassay 25 (OH) D
3With 1,25 (OH)
2D
3, with reflection kidney 1 α hydroxylase activity.
The kidney pathological observation:
Observation by light microscope: 6 week of irradiation back execution animal, get left kidney and go into to wash routine paraffin wax embedding, section, HE dyeing back observation by light microscope after the Susa immobile liquid is fixed 12 hours with 95% alcohol.
Electron microscope observation: get 3 1mm behind the sacrifice of animal immediately
3The right side cortex renis of size is gone into 2.5% glutaraldehyde and is made electron microscopic section after fixing and observe.
Kidney hydroxylase expression detects: (1) makes the kidney frozen section, and immunohistochemical method is observed kidney proximal tubule 1 α hydroxylase expression.(2) kidney homogenate detection method: kidney homogenate adds quantitative 25 (OH) D
3, detect 1,25 (OH)
2D
3The product level.
3. bone mineral content damage check:
Bone densitometry: get fourth lumbar vertebra, the right side femur is rejected soft tissue, the distilled water flushing ossis, and 120 ℃ of dryings 1 hour, the density of solid instrument is measured bone density;
The bone morphometry is measured: 14d before putting to death, 4d be lumbar injection quadracycline 30mg/kg body weight respectively.Get the left side femur upper segment, reject soft tissue, PBS washes ossis.Million`s is liquid-solid to decide 72h (4 ℃), and every 24h changes liquid once.The dehydration of ethanol gradient, methyl methacrylate infiltration embedding.Sclerosis back undecalcified microtome is cut 5 μ m, two kinds of sections of 15 μ m.Sheet is observed under fluorescent microscope after with the neutral resins mounting, thin slice spent glycol ether acetic acid esters takes off to be moulded, make morphometric and stereologic analysis after the dyeing of 2% toluidine blue, measure TBV, average bone trabecula thickness, average bone trabecula spacing, the terminal ratio of node, bone formation surface, bone resorption surface, mineralising deposition.
The present invention uses with the fixing kidney that exposes of kidney district irradiation mould
137Each 10-50Gy of Cs gamma-rays once irradiating bilateral kidney causes the kidney radiation insult, shows that urine protein obviously increases, and the urine β2Wei Qiudanbai increases, and the serum creatinine clearance rate reduces 1/3-1/2.Because kidney 1 α hydroxylase activity is subjected to press down serum 1,25 (OH)
2D
3Level is obviously low, descends nearly 1/2.The obvious change of bone mineral content structure took place after February, showed as bone density decline, TBV minimizing, the broadening of bone trabecula average headway, node end than following degradation.The present invention passes through surgical exposure bilateral kidney, and blocks kidney tissue in addition with stereotype, makes kidney irradiation accurate positioning, and has reduced the radioactive dose of kidney surrounding tissue to greatest extent.Cause the foundation of bone mineral content damage method by the radiation exposure kidney, for the fundamental research of bone mineral content metabolism provides new technological means with relevant control drug research.
Description of drawings
Fig. 1 is the pathological change of irradiation back kidney
Wherein A is normal mesangial cell, and B is a matter procollagen deposition between the irradiation metanephros, and C is normal kidney proximal tubule mitochondria, and D is the kidney proximal tubule mitochondrial swelling of irradiation back.
Embodiment
Embodiment 1
15Gy radiation gamma both sides kidney is to the influence of bone mineral content metabolism:
Experimental subjects: 20 of 3 monthly age male SD rats are divided into two groups of A, B, 10 every group at random.The A group is group in contrast, and B organizes as experimental group.
Operating process:
Anesthesia and kidney expose: each treated animal is weighed, lumbar injection 11% Ethylurethanm 0.5ml/100g body weight+ketalar 0.2ml/100g body weight.Pick hair, the routine disinfection drape.The rat right arm reclining, the stringer otch at 0.5cm place, the greater psoas muscle outside from the downward work of rib lower edge one long 1cm.Carefully open the abdominal cavity.Touch left kidney with hand at body surface, be pushed into it external through otch gently.Physiological saline gauze covering protection.The animal left lateral position exposes the right side kidney then.
Animal is fixed: crouch in mould in the animal right side, will reach four limbs end to end and fix.Animal kidney is coincide with the hole of stereotype to be aimed at.
Irradiation: with the B treated animal that fixes (the A group except that this step and B organize different, all the other are identical) put into the irradiation chamber of irradiation system, open radiation source, give pair each 15Gy radiation gamma of kidney respectively.
The irradiation aftertreatment: kidney is put back to the abdominal cavity, and layering is sewed up, and lumbar injection penicillin is anti-infective before the abdomen of pass.The rat that does not revive places warm place.Give normal drinking-water, diet, illumination after reviving.
Renal function and bone metabolism detection method are with above-mentioned technical scheme.
Experimental result shows that urine amount each time period after irradiation of A, B two treated animals do not have marked change substantially.But along with the prolongation of time, the basic no change of serum creatinine clearance rate of A treated animal, and the serum creatinine clearance rate of B treated animal descends gradually, the irradiation that kidney is described causes the reduction of glomerular filtration function.Compare with the A group, the urine β2Wei Qiudanbai/UCr value of B treated animal obviously raises, and illustrates that ray causes renal tubular function damage to a certain degree.The visible glomerular basement membrane of scanning electron microscope is irregular change, and capillary lumen obviously enlarges, and the interior visible migration inflammatory cell of ECS also has a large amount of procollagen depositions, and the renal tubular cell number reduces, the obvious swelling of cell mitochondrial, and optical density obviously reduces.Confirm that the inventive method employing 15Gy gamma-rays local irradiation rat bilateral kidney can cause the carrying out property decline of glomerulus, renal tubular function, so that the generation of chronic renal failure.Above-mentioned irradiation can influence the bone mineral content metabolism by several aspects to the damage of kidney, as Ca, P makes blood Ca through excessive the losing of urine, blood P loss of equilibrium, the destruction of nearly bent renal cells is reduced the necessary 1 α hydroxylase of synthesizing activity vitamin D, and Secondary cases parathyroid hormone (PTH) secretion increases the enhancing that can cause bone resorption.Bone density and bone morphometry result have confirmed the change of bone mineral content metabolism.Compare with the A group, the lumbar vertebrae of B treated animal, femur density all descend to some extent, and is wherein more obvious with lumbar vertebrae again.The TBV of irradiation animal reduces, the broadening of bone trabecula average headway, and node is terminal than reducing, and the bone formation surface reduces, and average bone trabecula spacing, bone resorption surface increase, the decline of mineralising deposition.The kidney electron microscopic observation is seen, the gap fibroplasia of B group glomerular basement membrane, and the proximal convoluted tubule mitochondrial swelling, electron density reduces, even has the cavity sample to change; The A group does not have the fibroplasia phenomenon is arranged, and the densification of proximal convoluted tubule mitochondria is clear, and electron density is higher, changes such as no swelling.Table 1 is an animal used as test urine protein result of variations.Table 2 is serum creatinine clearance rate (%).Table 3 records renal function index when being the experiment end.Blood urine routine biochemistry index when table 4 is the experiment end.Blood 25 (OH) D when table 5 is the experiment end
3, 1,25 (OH)
2D
3, PTH and urine PYD/ creatinine result.Table 6 is lumbar spine bmd and constituent analysis result.Table 7 is femoral bmd and constituent analysis result.Table 8 is bone norphometry results.Table 9 is bone mineralising deposition results.Table 10 is hydroxylase activities.Table 1
Group irradiation back 4 all 6 weeks of 2 weeks
Normal group (A) 0.895 ± 0.123 1.017 ± 0.189 0.823 ± 0.038
Irradiation group (B) 1.727 ± 0.378
*1.728 ± 0.169
*1.983 ± 0.583
*Annotate: in irradiation 2,4,6 weeks of back, compare the irradiation group with normal group
*P＜0.05,
*P＜0.01 table 2
First all second weeks of group
A 66.440±10.76 56.69±4.96
B 32.23 ± 11.75
*20.46 ± 5.34
*Annotate: compare the irradiation group with normal group
*P＜0.2 (no statistics difference),
*P＜0.01 table 3
Group blood urea nitrogen serum creatinine urine beta-2 microglobulin
(mmol/L) (mmol/L) (g/mL)
A 6.19±0.65 48.19±2.12 0.28±0.07
B 10.91±1.15
* 70.31±7.18 0.36±0.08
*
One-way analysis of variance,
*P＜0.05.(mean ± s, n=10) table 4
Group blood Ca blood P blood ALP urine Ca urine P
(mmol/L) (mmol/L) (U/L) (mmol/L) (mmol/L)
A 2.87±0.02 1.87±0.15 123.40±18.64 3.79±2.46 5.46±3.98
B 2.79±0.07 1.71±0.13 140.70±31.60
* 6.03±2.07
* 6.49±3.61
*
One-way analysis of variance,
*P＜0.05.(mean ± s, n=10) table 5
25 (OH) D
31,25 (OH)
2D
3PTH PYD/ creatinine
Group
(ng/mL) (pg/mL) (ng/dl) (nmol/mmol)
A 9.76±1.19 52.79±18.21 17.55±4.50 49.27±9.69
B 10.04±2.57 25.70±7.10
* 22.01±4.74
* 64.75±11.34
*
One-way analysis of variance,
*P＜0.05.(mean ± s, n=10) table 6
Group BMD (g/cm
2) dry weight (g) ash heavy (g)
A 0.34±0.02 0.30±0.03 0.21±0.02
B 0.30±0.03
* 0.27±0.02
* 0.19±0.01
*
One-way analysis of variance,
*P＜0.05.(mean ± s, n=10) table 7
Group BMD dry weight ash is heavy
(g/cm
3) (g) (g)
A 1.24±0.03 0.79±0.07 0.52±0.03
B 1.21±0.03
* 0.74±0.04
* 0.47±0.04
*
One-way analysis of variance,
*P＜0.05 (mean ± s, n=10) table 8
Group TBV bone trabecula average thickness bone trabecula average headway
(％) (μm) (μm)
A 25.19±2.67 35.51±5.56 110.21±11.27
B 21.79±1.84* 29.57±4.47* 128.48±24.60
One-way analysis of variance,
*P＜0.05.(mean ± s, n=10) table 9
Group MAR (μ m/d)
A 4.34±0.52
B 2.80±0.40
*
One-way analysis of variance,
*P＜0.05.(mean ± s, n=10) table 10
Group enzymatic activity (Pg/200mg/20min)
A 108.37±47.15
B 65.57±5.32
*
One-way analysis of variance,
*P＜0.05.(mean±s，n＝5)
Claims (2)
1, a kind of radiation kidney bone mineral content damage detecting method, it is characterized in that utilizing radiation exposure to cause kidney injury after, detect the change of bone mineral content amount and matter, set up radiation renal osteodystrophy detection method.
2, by the described radiation kidney of claim 1 bone mineral content damage detecting method, it is characterized in that being undertaken by following step,
1) accurate positioning experiment animal irradiated site carries out
137The Cs radiation gamma measures and is 10-50Gy.
2) injury of kidney detects, comprise blood urea nitrogen, creatinine dynamic monitoring, urine amount, urine protein, UCr clearance rate, the dynamic monitoring of urine β2Wei Qiudanbai, VD detects and microscope, electron microscope observation and the detection of kidney hydroxylase expression are learned by kidney trouble Ricoh.
3) bone mineral content damage check comprises bone densitometry and bone morphometry mensuration
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101027556B (en) * | 2004-06-07 | 2013-03-13 | 儿童医院医疗中心 | Method for the early detection of renal disease and injury |
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2002
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101027556B (en) * | 2004-06-07 | 2013-03-13 | 儿童医院医疗中心 | Method for the early detection of renal disease and injury |
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